NO154814B - PROCEDURE FOR IMMUNOLOGICAL DETERMINATION. - Google Patents
PROCEDURE FOR IMMUNOLOGICAL DETERMINATION. Download PDFInfo
- Publication number
- NO154814B NO154814B NO844764A NO844764A NO154814B NO 154814 B NO154814 B NO 154814B NO 844764 A NO844764 A NO 844764A NO 844764 A NO844764 A NO 844764A NO 154814 B NO154814 B NO 154814B
- Authority
- NO
- Norway
- Prior art keywords
- ligand
- enzyme
- receptor
- solution
- measurement
- Prior art date
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- 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
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- 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
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- 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
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Description
Foreliggende oppfinnelse vedrører en ny fremgangsmåte The present invention relates to a new method
for bestemmelse av immunologisk aktive materialer. for the determination of immunologically active materials.
I de siste årene er det utviklet mange analytiske systemer basert på kompetitiv protein-bindingsanalyse (også kalt metningsanalyse). In recent years, many analytical systems have been developed based on competitive protein binding analysis (also called saturation analysis).
Uttrykkene "metningsanalyse" og "kompetitiv protein-bindingsanalyse", som er synonyme, refererer til analytiske systemer som anvendes for bestemmelse av immunologisk aktive materialer (ligander). Resultatene av disse bestemmelsene i biologiske væsker anvendes i medisinsk og veterinærmedisinsk diagnose. Diagnosen avhenger av om mengden av substansen som skal bestemmes er normal eller patologisk. Det analytiske prinsippet er f.eks. basert på konkurransen mellom en ligand og en markert ligand som et felles spesifikt bindingsmiddel (reseptor) som illustert i den følgende reaksjonsligningen: The terms "saturation assay" and "competitive protein binding assay", 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 used in medical and veterinary medical diagnosis. The diagnosis depends on whether the amount of the substance to be determined is normal or pathological. The analytical principle is e.g. based on the competition between a ligand and a labeled ligand as a common specific binding agent (receptor) as illustrated in the following reaction equation:
Den primære reaksjonen er en kombinasjon av et molekyl av ligand med et molekyl av reseptor som danner et bi-molekylært ligand-reseptor-kompleks (1). En ytterligere reaksjon forårsakes ved addisjon av merket ligand som på lignende måte forbindes med reseptoren under dannelse av et merket ligand/reseptor-kompleks (2). I metningsanalyse er konsentrasjonene av reseptoren og den merkede liganden konstant. Reseptorkonsentrasjonen er begrenset slik at den merkede liganden er i overskudd i forhold til reseptoren. Under disse betingelser forårsaker addisjon av liganden konkurranse mellom ligand og markert ligand i binding til reseptoren. The primary reaction is a combination of a molecule of ligand with a molecule of receptor forming a bi-molecular ligand-receptor complex (1). A further reaction is caused by the addition of labeled ligand which similarly binds to the receptor forming a labeled ligand/receptor complex (2). In saturation analysis, the concentrations of the receptor and the labeled ligand are constant. The receptor concentration is limited so that the labeled ligand is in excess in relation to the receptor. Under these conditions, addition of the ligand causes competition between ligand and labeled ligand in binding to the receptor.
Derfor senker en økning av ligand-konsentrasjonen mengden av merket ligand/reseptor-kompleks. Målingsprinsippet er basert på bestemmelse av andel av totalt merket ligand bundet til reseptoren. Denne andelen er omvendt proporsjonal med mengden av ligand som tilsettes reaksjonsblandingen fra prøven som skal måles eller fra standarden som brukes ved målingen. Reduksjonen i konsentrasjonen av merket ligand/reseptor-kompleks eller økningen i konsentrasjon av merket ligand i reaksjonsblandingen kan brukes for å bestemme ligandkonsentrasjonen. Therefore, increasing the ligand concentration lowers the amount of labeled ligand/receptor complex. The measurement principle is based on determining the proportion of total labeled ligand bound to the receptor. This proportion is inversely proportional to the amount of ligand added to the reaction mixture from the sample to be measured or from the standard used in the measurement. The decrease in the concentration of labeled ligand/receptor complex or the increase in concentration of labeled ligand in the reaction mixture can be used to determine the ligand concentration.
Følsomheten til metningsanalysen avhenger av bruken av en reseptor som har en meget høy affinitet til liganden og merket ligand. Dertil avhenger følsomheten også av anvendelse av en markør som kan påvises i meget lav konsentrasjon. The sensitivity of the saturation assay depends on the use of a receptor that has a very high affinity for the ligand and labeled ligand. In addition, the sensitivity also depends on the use of a marker that can be detected in a very low concentration.
Metningsanalysens spesifisitet avhenger av reseptor-kapasiteten til utelukkende å binde ligand og merket ligand i en kompleks blanding av forskjellige molekyler. The specificity of the saturation assay depends on the receptor capacity to exclusively bind ligand and labeled ligand in a complex mixture of different molecules.
Metningsanalyse er blitt anvendt ved bruk av mange forskjellige typer teknikker, hvor forskjellen primært avhenger av typen merking som brukes. Generelt klassifiseres disse teknikkene ved å anvende omfattende titler angående "radioaktiv måling" eller "ikke-radioaktiv måling", og avhenger av om en radioaktiv tracer brukes som markør. Saturation analysis has been applied using many different types of techniques, the difference primarily depending on the type of labeling used. In general, these techniques are classified using broad headings regarding "radioactive measurement" or "non-radioactive measurement", depending on whether a radioactive tracer is used as a marker.
Radioaktiv måling har blitt anvendt i større utstrekning enn ikk-radioaktive målinger. Radioaktiv måling kan videre klassifiseres som radioaktiv-immuno-måling eller radioaktiv- reseptor-måling, avhengig av typen reseptor som benyttes i målingen. I radioaktiv-immuno-måling brukes et antistoff som spesifikt binder ligand og merket ligand. Alternativt vedrører radioaktiv-reseptor-analyse bruken Radioactive measurement has been used to a greater extent than non-radioactive measurements. Radioactive measurement can further be classified as radioactive-immuno-measurement or radioactive-receptor measurement, depending on the type of receptor used in the measurement. In radioactive immunoassays, an antibody is used that specifically binds ligand and labeled ligand. Alternatively, radioactive receptor analysis relates to its use
av hvilken som helst annen type biologisk reseptor som på lignende måte spesifikt vil binde liganden og den merkede liganden. of any other type of biological receptor that will similarly specifically bind the ligand and the labeled ligand.
I alle radioaktive målingsteknikker er det vesentlig å adskille fysikalsk den bundne fraksjon (1 og 2 ifølge likningen som er angitt tidligere) fra den ikke-bundne fraksjon (3 og 4 i den tidligere angitte ligning) i reaksjonsblandingen. En indeks på ligandkonsentrasjonen kan deretter oppnås ved å telle disse fraksjoner i et radioaktivt telleverk og sammenligne tellingene som oppnås for de ukjente prøvene med dem som oppnås for egnede standard-ligand-prøver som underkastes den samme målingen. Mange forskjellige og avvikende metoder er blitt beskrevet for adskillelse av de bundne og frie fraksjonene i den radioaktive målingsreaksjonsblandingen, og disse benytter teknikker som gelfiltrering, absorpsjon-og ionebytterkromatografi, fraksjonert felling, fast fase eller elektroforese. In all radioactive measurement techniques, it is essential to physically separate the bound fraction (1 and 2 according to the equation previously stated) from the non-bound fraction (3 and 4 in the previously stated equation) in the reaction mixture. An index of the ligand concentration can then be obtained by counting these fractions in a radioactive counter and comparing the counts obtained for the unknown samples with those obtained for suitable standard ligand samples subjected to the same measurement. Many different and divergent methods have been described for the separation of the bound and free fractions in the radioactive measurement reaction mixture, and these use techniques such as gel filtration, absorption and ion exchange chromatography, fractional precipitation, solid phase or electrophoresis.
Etter utviklingen av radioaktive målingsteknikker i metningsanalysen, er metoder som anvender ikke-radioaktive markører blitt utviklet. Metoder som anvender enzymer som markører er blitt vist. Disse metodene kan ha den fordel at fysikalsk adskillelse av bundne (1 + 2) og frie (3+4) fraksjoner av merket ligand ikke er nødvendig i måleprosedyren. Following the development of radioactive measurement techniques in saturation analysis, methods using non-radioactive markers have been developed. Methods using enzymes as markers have been demonstrated. These methods may have the advantage that physical separation of bound (1 + 2) and free (3 + 4) fractions of the labeled ligand is not necessary in the measurement procedure.
Når antistoffer binder ligand som er merket med et enzym, modifiseres aktiviteten av enzymet. Modifikasjonsgraden til enzymaktiviteten indikerer konsentrasjonen av den merkede liganden i den bundne fraksjonen, og gir derfor en indeks på ligandkonsentrasjonen i reaksjonsblandingen. When antibodies bind ligands labeled with an enzyme, the activity of the enzyme is modified. The degree of modification of the enzyme activity indicates the concentration of the labeled ligand in the bound fraction, and therefore provides an index of the ligand concentration in the reaction mixture.
Den kjemiske strukturen til ligand-enzym-komplekset er ekstremt vanskelig å klarlegge, og dette er en vesentlig ulempe ved enzym-immuno-målingen. Dette skyldes utvilsomt det store antall aminosyrekjeder som foreligger på enzym-overflaten ved kompleksering med liganden. Dette forårsaker store vanskeligeheter ved reproduksjon av ligand-enzymet i forskjellige fremstillinger av komplekset. The chemical structure of the ligand-enzyme complex is extremely difficult to clarify, and this is a major disadvantage of the enzyme-immuno-measurement. This is undoubtedly due to the large number of amino acid chains present on the enzyme surface when complexed with the ligand. This causes great difficulties in reproducing the ligand-enzyme in different preparations of the complex.
Den generelle mangel på kontroll over komplekserings-reaksjonen resulterer i tilknytningen av mange ligandmolekyler til et enzym-molekyl, skjønt det er sannsynlig at binding av bare noen få av disse ligandmolekylene ved antistoffer inngår i inhiberingen av enzymaktiviteten. Derfor resulterer ikke alle antistoff/merket antigen-forbindelser i en modifisering av enzymaktiviteten, hvilket derved senker teknikkens sensibilitet. The general lack of control over the complexation reaction results in the attachment of many ligand molecules to an enzyme molecule, although it is likely that binding of only a few of these ligand molecules by antibodies is involved in the inhibition of enzyme activity. Therefore, not all antibody/labeled antigen compounds result in a modification of the enzyme activity, which thereby lowers the sensitivity of the technique.
Protein-protein-interaksjonen mellom forskjellige anti-stoffmolekyler og antistoff og enzym-molekyler er en annen følge av antallet ligandmolekyler på enzymover-flaten. Protein-protein-interaksjonen økes videre hvis liganden også er et polypeptid. Således induserer enzym-molekylet et lokalt mikromiljø med høy proteinkonsentra-sjon. I slike situasjoner er det vist at proteinfelling opptrer, og som derved forårsaker tap av en av hovedfordelene ved enzym-immunomåling ved at separasjon av antistoffbundne og frie fraksjoner blir nødvendige. The protein-protein interaction between different antibody molecules and antibody and enzyme molecules is another consequence of the number of ligand molecules on the enzyme surface. The protein-protein interaction is further increased if the ligand is also a polypeptide. Thus, the enzyme molecule induces a local microenvironment with a high protein concentration. In such situations, it has been shown that protein precipitation occurs, which thereby causes the loss of one of the main advantages of enzyme-immunoassay in that separation of antibody-bound and free fractions becomes necessary.
Det er beskrevet en modifikasjon av enzymatisk immuno-måling som delvis overvinner disse problemene ved at liganden merkes med et detektormolekyl som har en lav molekylvekt. I denne målingen hindres antistoff-bindingen av merket«ligand-binding av et detektor-molekyl ved et annet antistoff som er spesifikt for detektormolekylet. Indeksgraden bestemmes ved konkurranse av binding av det frie ligand-detektormolekyl og detektor-molekyl-merket-enzym med detektor-molekyl-antistoff. Modifikasjonsgraden av enzymaktivitet bestemmes i normal enzymatisk immuno-måling, og indikerer konsentrasjonen av fritt ligand-detektor-molekyl som likeledes er en indikasjon på ligand-konsentrasjonen i reaksjonsblandingen. Fordelen ved denne teknikk er at et lite molekyl fremfor et enzym knyttes til liganden, hvilket derved muliggjør at den kjemiske strukturen til den merkede liganden kan bestemmes, og overvinner således mange av de ulemper som er knyttet til de tidligere beskrevne enzymatiske immuno-målinger. Imidlertid overvinner dette systemet bare ulemper ved den primære bindingsreaksjonen som innebefatter liganden og merket ligand, og overfører dem til detektorsystemet for -bestemmelse av graden av bundet antistoff og fritt antistoff fra fraksjoner i den merkede liganden. A modification of enzymatic immuno-measurement has been described which partially overcomes these problems by labeling the ligand with a detector molecule which has a low molecular weight. In this measurement, the antibody binding of the labeled "ligand" binding of a detector molecule is prevented by another antibody that is specific for the detector molecule. The degree of index is determined by competition of binding of the free ligand-detector molecule and detector-molecule-labeled-enzyme with detector-molecule-antibody. The degree of modification of enzyme activity is determined in normal enzymatic immuno-measurement, and indicates the concentration of free ligand detector molecule which is likewise an indication of the ligand concentration in the reaction mixture. The advantage of this technique is that a small molecule rather than an enzyme is attached to the ligand, which thereby enables the chemical structure of the labeled ligand to be determined, and thus overcomes many of the disadvantages associated with the previously described enzymatic immuno-measurements. However, this system only overcomes disadvantages of the primary binding reaction involving the ligand and labeled ligand and transfers them to the detector system for determining the degree of bound antibody and free antibody from fractions in the labeled ligand.
Ulempene ved de tidligere kjente metoder overvinnes ved foreliggende oppfinnelse, hvor et enzym-modifiseringsmiddel brukes som markør. The disadvantages of the previously known methods are overcome by the present invention, where an enzyme modifier is used as a marker.
Nærmere bestemt vedrører foreliggende oppfinnelse en fremgangsmåte for bestemmelse av et immunologisk aktivt materiale i en prøve, hvor prøven bringes i kontakt med en reseptor som spesifikt kan binde det immunologisk aktive materiale, og med det immunologisk aktive materiale merket med en substans som er i stand til å modifisere graden eller typen av enzymets aktivitet, og med et enzym eller et enzymsubstrat, og hvor graden eller typen av den resulterende enzymaktivitet måles og sammenlignes med resultatene erholdt fra en standard. More specifically, the present invention relates to a method for determining an immunologically active material in a sample, where the sample is brought into contact with a receptor that can specifically bind the immunologically active material, and with the immunologically active material labeled with a substance capable of to modify the degree or type of the enzyme's activity, and with an enzyme or an enzyme substrate, and where the degree or type of the resulting enzyme activity is measured and compared to the results obtained from a standard.
Uttrykkene "immunologisk aktivt materiale" eller "ligand" The terms "immunologically active material" or "ligand"
i denne beskrivelsen refererer til enhver immunologisk aktiv substans eller del av denne som er i stand til å bestemmes immunologisk, f.eks. ved å bruke metningsanalyse-teknikker. Det viktigste kravet er at det er en reseptor in this description refers to any immunologically active substance or part thereof capable of being determined immunologically, e.g. using saturation analysis techniques. The most important requirement is that there is a receptor
som spesifikt vil binde liganden. Når reseptoren er et antistoff, vil liganden være et hapten eller antigen, which will specifically bind the ligand. When the receptor is an antibody, the ligand will be a hapten or antigen,
slik at det spesifikke antistoff kan dannes. En ligand i denne sammenheng er et hapten når det bare vil gi antistoff-dannelse ved tilknytning til antigen. Alternativt er en ligand i denne forbindelse et antigen når det vil gi antistoffdannelse uten kjemisk modifisering. Liganden kan variere sterkt i molekylvekt, fra 100 - 1 000 000. Dette molekylvektsområdet er ikke begrensende i målingen såfremt reseptoren er i stand til spesifikt å binde liganden. so that the specific antibody can be formed. A ligand in this context is a hapten when it will only cause antibody formation when attached to antigen. Alternatively, a ligand in this connection is an antigen when it will produce antibody formation without chemical modification. The ligand can vary greatly in molecular weight, from 100 - 1,000,000. This molecular weight range is not limiting in the measurement provided the receptor is able to specifically bind the ligand.
liganden kan enten ha polymer- eller ikke-polymer struktur. Ved polymer-struktur vil liganden normalt være av biologisk opprinnelse, og kan klassifiseres som en nuklein-syre-polysakkarid og/eller polypeptid. Alternativt vil liganden, når den ikke er polymer, generelt ha en molekylvekt mindre enn 2 00 0 og kan ha ganske forskjellige strukturer, funksjonelle grupper og fysiologiske egenskaper. the ligand can either have polymeric or non-polymeric structure. In the case of a polymer structure, the ligand will normally be of biological origin, and can be classified as a nucleic acid polysaccharide and/or polypeptide. Alternatively, the ligand, when non-polymeric, will generally have a molecular weight of less than 2,000 and may have quite different structures, functional groups and physiological properties.
Ligander av særlig betydning som kan brukes i foreliggende oppfinnelse er aminer, aminosyrer, peptider, proteiner, lipoproteiner, glykoproteiner, steroler, steroider, lipoider, nukleinsyrer, mono- eller polysakkarider, alkaloider, vitaminer, droger, narkotika, antibiotika, metabolitter, pesticider, toksiner, industrielle bi-produkter, smaksmidler, hormoner, enzymer, koenzymer, cellulære eller ekstracellulære komponenter fra vev og isolerte antistoffer fra mennesker eller dyr. Målingen er imidlertid ikke begrenset til bare disse ligandene. Ligands of particular importance that can be used in the present invention are amines, amino acids, peptides, proteins, lipoproteins, glycoproteins, sterols, steroids, lipoids, nucleic acids, mono- or polysaccharides, alkaloids, vitamins, drugs, drugs, antibiotics, metabolites, pesticides, toxins, industrial by-products, flavourings, hormones, enzymes, coenzymes, cellular or extracellular components from tissues and isolated antibodies from humans or animals. However, the measurement is not limited to only these ligands.
Komponenter som umiddelbart egner seg for analyse i systemet er hepatitt-B overflate-antigen, ferritin, tumor antigener som CEA, Æ-føto protein, rheumatiod faktor, C-reaktive proteiner, immunoglobulin klasser IgG, IgM eller IgA, myoglobin, tyroide hormoner inklusive T_ og T^, insulin, steroide hormoner innebefattende testosteron eller estradiol, misbrukte droger innebefattende narkot-iske sovemidler som morfin, barbiturater, stimulerende midler som amfetamin, droger for behandling av epilepsi innebefattende difenyl-hdrantion og fenobarbital, krets-løps glykocytter som digoksin, vitaminer som vitamin og folinsyre. Videre antistoffer som umiddelbart er egnet for analyse i systemet er slike som forekommer ved infek-sjon av syfilis, gonoré, brucellosis, rubella og rheumat-isme. Components that are immediately suitable for analysis in the system are hepatitis B surface antigen, ferritin, tumor antigens such as CEA, Æ-feto protein, rheumatoid factor, C-reactive proteins, immunoglobulin classes IgG, IgM or IgA, myoglobin, thyroid hormones included T_ and T^, insulin, steroid hormones including testosterone or estradiol, drugs of abuse including narcotic hypnotics such as morphine, barbiturates, stimulants such as amphetamines, drugs for the treatment of epilepsy including diphenylhydranthion and phenobarbital, circulating glycosides such as digoxin, vitamins such as vitamin and folic acid. Furthermore, antibodies that are immediately suitable for analysis in the system are those that occur during infection with syphilis, gonorrhea, brucellosis, rubella and rheumatism.
Uttrykket "merket immunologisk aktivt materiale" eller "merket reseptor" i denne beskrivelse refererer til en ligand, analog av en ligand eller en del av denne, eller til en reseptor som er merket med et enzym-modifiseringsmiddel. En, flere enn en, og generelt mindre enn 100 molekyler av markør kan knyttes til en ligand eller et reseptormolekyl. Likeledes kan et, fler enn et, og normalt mindre enn 5 ligander eller reseptormolekyler knyttes til et markørmolekyl. Tilknytning av ekstra markørmolekyler til en ligand eller et reseptormolekyl øker generelt målings-følsomheten, forutsatt at de ekstra markørene ikke påvirker bindingen. The term "labeled immunologically active material" or "labeled receptor" in this specification refers to a ligand, analog of a ligand or part thereof, or to a receptor that is labeled with an enzyme-modifying agent. One, more than one, and generally less than 100 molecules of label can be attached to a ligand or a receptor molecule. Likewise, one, more than one, and normally less than 5 ligands or receptor molecules can be attached to a marker molecule. Attachment of additional marker molecules to a ligand or a receptor molecule generally increases measurement sensitivity, provided that the additional markers do not affect binding.
Tilknytning av et modifiseringsmolekyl til en ligand eller reseptormolekyl innebefatter dannelsen av inter-molekylære bindinger som i de fleste tilfeller, men ikke nødvendigvis, er av kovalent natur. Bindingen kan i noen tilfeller utføres i nærvær av et koplingsmiddel ved inn-føring av en bindingsgruppe mellom markøren og liganden eller reseptoren. Attachment of a modifier molecule to a ligand or receptor molecule involves the formation of inter-molecular bonds which in most cases, but not necessarily, are covalent in nature. In some cases, the binding can be carried out in the presence of a coupling agent by introducing a binding group between the marker and the ligand or receptor.
Det modifiserende molekyl kan knyttes direkte til liganden eller reseptormolekylet. Imidlertid kan det være ønskelig å innføre kjemiske broer av forskjellige lengder mellom modifiseringsmolekylet og liganden eller reseptormolekylene, avhengig av den spesifikke målingen som skal utføres. I noen tilfeller kan det også være en fordel å knytte modifiseringsmolekylet og liganden eller reseptormolekylene separat til det samme bæremolekylet, f.eks. et makromolekyl som et polypeptid eller et poly-sakkarid . The modifying molecule can be linked directly to the ligand or the receptor molecule. However, it may be desirable to introduce chemical bridges of different lengths between the modifier molecule and the ligand or receptor molecules, depending on the specific measurement to be performed. In some cases, it may also be advantageous to attach 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.
Uttrykket "reseptor" i denne beskrivelsen refererer seg til enhver substans som spesifikt kan binde liganden og merket ligand eller en del av denne. Generelt er den anvendte reseptor i målingen et spesifikt antistoff for liganden som dannes i blodet hos hvirveldyr etter injek-sjon av riktig hapten eller antigen. Alternativt kan reseptorer som opptrer i naturen også brukes i målingen. Denne siste gruppen innebefatter, men er ikke begrenset til dette, proteiner, nukleinsyrer og cellulære membraner. Slike reseptorer er blitt brukt i radioaktive måleteknikker for tyroksin, insulin, angiotensin og forskjellige s teroidhormoner. The term "receptor" in this specification refers to any substance that can specifically bind the ligand and labeled ligand or part thereof. In general, the receptor used in the measurement is a specific antibody for the ligand that is formed in the blood of vertebrates after injection of the correct hapten or antigen. Alternatively, receptors that occur in nature can also be used in the measurement. This last group includes, but is not limited to, proteins, nucleic acids and cellular membranes. Such receptors have been used in radioactive measuring techniques for thyroxine, insulin, angiotensin and various steroid hormones.
I tilfelle liganden er et antistoff, kan reseptoren være antigenet som brukes for å indusere dette antistoffet i et vertsdyr. I en annen utførelsesform kan reseptoren være et antistoff mot antistoffet som skal bestemmes. In the event that 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 can be an antibody against the antibody to be determined.
Det er ikke mulig å fastlegge med sikkerhet reseptorens virkemåte, som ved binding av den merkede liganden reduserer virkningen mellom enzym og modifiseringsmiddel. Den sannsynligste forklaringen er at enzymets affinitet til modifiseringsmiddelet nedsettes som følge av en størrelsesforandring og netto ladning hos reseptor/ligand-modifiseringskomplekset sammenlignet med den for ligand-modifiseringen alene. It is not possible to determine with certainty the mode of action of the receptor, which, by binding the labeled ligand, reduces the effect 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 compared to that of the ligand modifier alone.
Uttrykket "modifiseringsmiddel" i denne beskrivelsen refererer til enhver substans som er i stand til å inn-virke på et enzym slik at graden eller typen av enzymaktivitet modifiseres. Denne modifiseringen kan resultere i en inhibering, aktivering eller spesifisitetsforandring eller enhver annen egenskapsforandring hos enzymet som er påviselig enten direkte eller indirekte ved en forandring i enzymaktiviteten eller i måten av aktivitet, f.eks. en forandring i reaksjonsbetingelser som ko-faktor-behov eller pH-optimum, i kinetiske egenskaper eller i aktiverings-energi. Modifiseringsmiddelet kan variere i størrelse fra et lite molekyl til et makromolekyl, og dets innvirkning på et enzymmolekyl kan enten være reversibelt eller ir-reversibelt avhengig av om den inter-molekylære assosiasjon er av ionisk eller kovalent natur. The term "modifying agent" in this description refers to any substance capable of acting on an enzyme so that the degree or type of enzyme activity is modified. This modification may result in an inhibition, activation or specificity change or any other property change of the enzyme which is detectable either directly or indirectly by a change in the enzyme activity or in the mode of activity, e.g. a change in reaction conditions such as co-factor requirement or pH optimum, in kinetic properties or in activation energy. The modifier can vary in size from a small molecule to a macromolecule, and its effect on an enzyme molecule can be either reversible or irreversible depending on whether the inter-molecular association is ionic or covalent in nature.
Målingsømfintligheten er bl.a. avhengig av reseptorens affinitet overfor liganden og modifiseringsmiddelets evne til å fremkalle en forandring i grad eller type av enzymaktivitet. Fortrinnsvis er modifikasjonen av graden eller typen av enzymaktivitet oppnåelig med en minimal konsentrasjon av modifiseringsmiddelet. Jo nærmere denne konsentrasjonen ligger enzymets på molekylær basis, desto større vil måieømfintligheten være. The measurement sensitivity is i.a. depending on the affinity of the receptor towards the ligand and the ability of the modifier to induce a change in the degree or type of enzyme activity. Preferably, the modification of the degree or type of enzyme activity is achievable with a minimal concentration of the modifying agent. The closer this concentration is to the enzyme's on a molecular basis, the greater the molecular sensitivity will be.
Fortrinnsvis vil modifiseringsmiddelet være en enzyminhibitor som ved innvirkning på et enzym vil inhibere dets aktivitet. Virkningsmåten av inhibitoren kan være kompetitiv, non-kompetitiv, ikke-kompetitiv, allosterisk eller en kombinasjon av to eller flere av disse måtene. Fortrinnsvis bør inhibitoren ha en inhiberingskonstant (nødvendig inhibitor-konsentrasjon for 50% inhibering av Preferably, the modifying agent will be an enzyme inhibitor which, when acting on an enzyme, will inhibit its activity. The mode of action of the inhibitor may be competitive, non-competitive, non-competitive, allosteric or a combination of two or more of these modes. Preferably, the inhibitor should have an inhibition constant (required inhibitor concentration for 50% inhibition of
_ 3 _ 3
enzymsystemet) under 10 mol pr. liter, avhengig av den foreliggende måling. Særlig foretrukket er inhiberings--15 -5 the enzyme system) below 10 mol per litres, depending on the current measurement. Particularly preferred is inhibition -15 -5
konstanten mellom 10 og 10 the constant between 10 and 10
Ethvert enzym kan brukes i målingen, forutsatt at det eksisterer et modifiseringsmiddel som spesifikt vil modifisere enzymaktiviteten på den overfor beskrevne måte. Aktuelle enzymer er stabile, lett tilgjengelige ved lave omkostninger og har et høyt o<y>erføringstall og et enkelt utført målesystem. Fortrinnsvis er overførings-tallet (molekylprodukt som dannes pr. enzymmolekyl pr. minutt) over 100, avhengig av det spesifikke forsøk som utføres. Fortrinnsvis er overføringstallet så høyt som mulig, og minst 20 0. Any enzyme can be used in the measurement, provided that a modifier exists which will specifically modify the enzyme activity in the manner described above. Current enzymes are stable, easily available at low cost and have a high conversion rate and a simple measurement system. Preferably, the transfer number (molecular product formed per enzyme molecule per minute) is above 100, depending on the specific experiment being performed. Preferably, the transmission number is as high as possible, and at least 200.
Enzymmodifiseringssystemet som er spesielt egnet for den foreliggende oppfinnelse er dihydrofolat reduktase/metotreksat, dihydrofolat reduktase/4-aminopterin, dihydrofolat reduktase/andre spesifikke inhibitorer for dette enzymet, j3 -glukoronidase/4-deoksy-5-amino-glutarsyre eller derivater derav, biotin inneholdende enzymer/avidin The enzyme modification system which is particularly suitable for the present invention is dihydrofolate reductase/methotrexate, dihydrofolate reductase/4-aminopterin, dihydrofolate reductase/other specific inhibitors of this enzyme, j3-glucoronidase/4-deoxy-5-amino-glutaric acid or derivatives thereof, biotin containing enzymes/avidin
som karboksylase/avidin, chymotrypsin/TPCK (TPCK har formel: as carboxylase/avidin, chymotrypsin/TPCK (TPCK has formula:
X -cystationase/proparylglysin, alanin racemase/trifluoralanin, tryptofanase/trifluoralanin, tryptofan syntetase/ trifluoralanin, p> -cystationase/trifluoralanin, pyruvat-glutamat transaminase/trifluoralanin, melkesyre oksydase/ 2-hydroksy-3-butynsyre, monoamin oksydase/N,N-dimetyl propargylamin og diaminoksydase/H2N-CH2-C=C-CH2-NH2. X -cystathionase/proparylglycine, alanine racemase/trifluoroalanine, tryptophanase/trifluoroalanine, tryptophan synthetase/trifluoroalanine, p>-cystathionase/trifluoroalanine, pyruvate-glutamate transaminase/trifluoroalanine, lactic acid oxidase/ 2-hydroxy-3-butyric acid, monoamine oxidase/N, N-dimethyl propargylamine and diamine oxidase/H2N-CH2-C=C-CH2-NH2.
Bestemmelse av enzymaktivitet kan utføres ved å regulere direkte eller indirekte substratforbruket eller produk-sjonen av produktet ved passende pH og temperatur ved anvendelse av påvisningssystemer som innebefatter kolori-metri, spektrofotometri, fluorospektrofotometri, gasio-metri, termometri (varmeutvikling) eller scintillasjons-telling. Determination of enzyme activity can be performed by directly or indirectly regulating substrate consumption or product production at appropriate pH and temperature using detection systems including colorimetry, spectrophotometry, fluorospectrophotometry, gasiometry, thermometry (heat generation) or scintillation counting.
For å øke systemets ømfintlighet er det mulig å bruke bioluminescens og enzymcykliseringsteknikker, f.eks. de teknikker som er beskrevet av J.Lee et al i "Liquid Scintillation Counting: Recent Developments", Stanley To increase the sensitivity of the system, it is possible to use bioluminescence and enzyme cyclization techniques, e.g. the techniques described by J. Lee et al in "Liquid Scintillation Counting: Recent Developments", Stanley
P.E. and Scoggins, B.A., Academic Press, New York, p.403 P.E. and Scoggins, B.A., Academic Press, New York, p.403
og Lowry O.H. et al i J. Biol. Chem. 236, p. 2746-2755. and Lowry O.H. et al in J. Biol. Chem. 236, pp. 2746-2755.
Ifølge foreliggende oppfinnelse kan en merket ligand According to the present invention, a labeled ligand can
brukes for bestemmelse av tilstedeværelsen av en ligand i en ukjent prøve ved den stimulante eller etterfølgende tilsitning av merket ligand og den ukjente prøven til et vandig medium ved egnet pH som inneholder en spesifikk reseptor for liganden og den merkede liganden. Etter en passende inkuberingstid bestemmes fordelingen av reseptoren bundet til ligand og merket ligand ved tilsetning av enzym og substrater. Reseptoren som er spesifikk for liganden, binder også den merkede liganden, og reduserer derved innvirkningen mellom modifiseringsmidlet og enzymet, og nedsetter derved modifiseringen av enzymaktiviteten. Tilsetning av ligand til prøven resulterer i konkurranse med den merkede ligand i binding til reseptor, og øker derved konsentrasjonen av fri merket ligand i målingen. Innvirkningen mellom ligandmodifiserings-middel og enzym økes, og enzymaktiviteten blir igjen påvirket. Modifiseringen av enzymaktiviteten er derfor en funksjon av ligandkonsentrasjonen i målingen, og bevirkes av ubundet merket ligand. Følgelig er forskjellen mellom den resulterende enzymaktivitet og den som beholdes uten ligand, et mål for konsentrasjonen av ligand i den ukjente prøven. is used for the determination of the presence of a ligand in an unknown sample by the stimulating or subsequent addition of the labeled ligand and the unknown sample to an aqueous medium at a suitable pH containing a specific receptor for the ligand and the labeled ligand. After a suitable incubation time, the distribution of the receptor bound to ligand and labeled ligand is determined by addition of enzyme and substrates. The receptor specific for the ligand also binds the labeled ligand, thereby reducing the interaction between the modifier and the enzyme, thereby reducing the modification of the enzyme activity. Addition of ligand to the sample results in competition with the labeled ligand in binding to the receptor, thereby increasing the concentration of free labeled ligand in the measurement. The interaction between ligand modifier and enzyme is increased, and the enzyme activity is again affected. The modification of the enzyme activity is therefore a function of the ligand concentration in the measurement, and is effected by unbound labeled ligand. Consequently, the difference between the resulting enzyme activity and that retained without ligand is a measure of the concentration of ligand in the unknown sample.
En av hovedfordelene ved denne metoden er at adskillelsen One of the main advantages of this method is that the separation
av bundne og frie fraksjoner er unødvendige i fremgangs- of bound and free fractions are unnecessary in progress
måten. the way.
Denne målingen er selvfølgelig ikke begrenset til bestemmelse av haptener og antigener som liganden, men kan også tilpasses for identifisering og måling av antistoffer som liganden. Dette kan f.eks. utføres ved å merke antistoffet med et enzymmodifiseringsmiddel, og måle graden av enzymaktivitetsmodifisering etter inkubering av merket antistoff og prøve-antistoff med en begrensende konsentrasjon av antigen eller hapten. Modifiseringen av enzymaktivitet vedrører prøve-antistoff-konsentrasjonen. This measurement is of course not limited to the determination of haptens and antigens as the ligand, but can also be adapted for the identification and measurement of antibodies as the ligand. This can e.g. is performed by labeling the antibody with an enzyme modifier, and measuring the degree of enzyme activity modification after incubation of the labeled antibody and sample antibody with a limiting concentration of antigen or hapten. The modification of enzyme activity relates to the sample-antibody concentration.
Reagenser til bruk ved fremgangsmåten ifølge foreliggende oppfinnelse, også beskrevet i utlegningsskrift nr. 152.955, fremstilles på følgende måte: Reagents for use in the method according to the present invention, also described in explanatory document no. 152,955, are prepared in the following way:
Fremstilling av " amino- digoksin". Preparation of "amino-digoxin".
En suspensjon av 156 mg (0,2 mmol) digoksin i 5 ml absolutt etanol ble tilsatt 10 ml 0,2 M natriummeta-periodat under røring. Blandingen ble homogen etter 10 minutter, og så ble fellingen langsomt dannet. Etter 2 timer ble 5 ml vann pluss 5 ml etanol tilsatt. 122 ul (2,2 mmol) etylenglykol ble tilsatt etter ytterligere 30 minutter, og et tett nytt presipitat begynte straks å dannes. Etter 80 minutters røring ble 133 ul (2,0 mmol) etylendiamin tilsatt, og den resulterende pH på 11,0 ble justert til 9,5 med 0,1 M HC1, og reaksjonsblandingen fikk stå ved en romtemperatur i 18 timer. pH forandret seg ikke i dette tidsrommet. A suspension of 156 mg (0.2 mmol) of digoxin in 5 ml of absolute ethanol was added to 10 ml of 0.2 M sodium meta-periodate with stirring. The mixture became homogeneous after 10 minutes, and then the precipitate slowly formed. After 2 hours, 5 ml of water plus 5 ml of ethanol were added. 122 µl (2.2 mmol) of ethylene glycol was added after another 30 minutes, and a dense new precipitate immediately began to form. After 80 minutes of stirring, 133 µl (2.0 mmol) of ethylenediamine was added, and the resulting 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 hours. The pH did not change during this time.
151,4 mg (4,0 mmol) natriumborhydrid ble så tilsatt, og blandingen rørt i 3 1/2 time. pH ble så justert fra 10,5 til 6,5 med 1 M maursyre (ca. 3 ml) og TLC av denne blandingen viste en enkel hovedflekk med en Rf lik 0,15 (kiselgel på aluminium utviklet i butanol:iseddik:vann/ 4:1:1). Digoksin hadde en Rf på 0,7 ved utvikling i 151.4 mg (4.0 mmol) of sodium borohydride was then added and the mixture stirred for 3 1/2 hours. The pH was then adjusted from 10.5 to 6.5 with 1 M formic acid (ca. 3 mL) and TLC of this mixture showed a single major spot with an Rf equal to 0.15 (silica gel on aluminum developed in butanol:glacial acetic acid:water/ 4:1:1). Digoxin had an Rf of 0.7 when developed i
samme system. same system.
Løsningsmidlene ble avdampet nesten til tørrhet på en rotasjonsfordamper under vakuum ved anvendelse av et vann-bad på 60°C. De siste få ml vann ble fjernet ved tilsetning av 95% etanol (3 x 20 ml) og gjentagelse av inndampningen som ovenfor. The solvents were evaporated to near dryness on a rotary evaporator under vacuum using a water bath at 60°C. The last few ml of water were removed by adding 95% ethanol (3 x 20 ml) and repeating the evaporation as above.
Det resulterende blekgule, faste stoff ble ekstrahert tre ganger med absolutt etanol, og disse kombinerte ekstraktene konsentrert i ca 4 ml og sentrifugert for å adskille en liten mengde salt, som ble kastet. Den blekgule supernatantløsningen ble inndampet til tørrhet under en strøm av tørt nitrogen, og ga en gul olje som viste samme Rf på TLC som reaksjonsblandingen som er beskrevet ovenfor. Produktet ble vist å inneholde en ■ fri aminogruppe ved å omsette den med "Fluram" (4-fenyl-spiro(fluran-2(3H),1<1->ftalan)-3,3<1->dion) under dannelse av en sterkt fluoriserende forbindelse. The resulting 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 supernatant 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 described above. The product was shown to contain a ■ free amino group by reacting it with "Fluram" (4-phenyl-spiro(flurane-2(3H),1<1->phthalan)-3,3<1->dione) forming of a highly fluorescent compound.
Produktet hadde et spektrum i konsentrert svoverlsyre i likhet med oligoksinets med absorpsjonstopper ved 385 og 495 mu. Produktet hadde også en sterk affinitet for spesifikke antisera (kanin) for digoksin. Den sannsynligste struktur for det isolerte produkt er den følgende: The product had a spectrum in concentrated sulfuric acid similar to that of oligoxine with absorption peaks at 385 and 495 mu. The product also had a strong affinity for specific antisera (rabbit) for digoxin. The most likely structure for the isolated product is the following:
Fremstilling av metotreksat- aminodigoksin- konjugat. Preparation of methotrexate-aminodigoxin conjugate.
11 mg metotreksat ble oppløst i 5 ml I^o og justert til pH 6,5. 10 mg "aminodigoksin" ble oppløst i denne løsningen, og volumet justert til 20 ml med vann. pH 11 mg of methotrexate was dissolved in 5 ml of I20 and adjusted to pH 6.5. 10 mg of "aminodigoxin" was dissolved in this solution, and the volume adjusted to 20 ml with water. pH
ble justert til 6,5 og 484 mg N-etyl,N'1 -(3-dimetylamino)-propyl-karbodiimid-hydroklorid oppløst i 5 ml vann ble satt til reaksjonsblandingen. pH ble holdt på 6,2 i 24 timer ved romtemperatur. Konjugatet ble renset på kisel-gelkolonne ved anvendelse av 3% ammoniusitrat som løsningsmiddel. Fraksjoner som inneholdt det ønskede produkt ble slått sammen og vist å ha samme dobbelte evne til å binde sterke antisera (kanin) spesifikke for digoksin, og inhiberte også sterkt enzymet dihydrofolatreduktase (kyllinglever). Den sannsynligste struktur for metotreksat-aminodigoksin-konjugatet er den følgende: was adjusted to 6.5 and 484 mg of N-ethyl,N'1-(3-dimethylamino)-propylcarbodiimide hydrochloride dissolved in 5 ml of water 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 3% ammonium citrate as solvent. Fractions containing the desired product were pooled and shown to have the same dual ability to bind strong antisera (rabbit) specific for digoxin, and also strongly inhibited the enzyme dihydrofolate reductase (chicken liver). The most likely structure for the methotrexate-aminodigoxin conjugate is the following:
Fremstilling av metotreksat- humant serumalbumin- kojugat. Preparation of methotrexate human serum albumin conjugate.
45 mg metotreksat ble oppløst i 1,0 ml N,N-dimetyl-formamid og 25 mg N-hydroksysuccinimid ble tilsatt. 41 mg N,N-dicykloheksyl-karbodiimid ble oppløst, og blandingen holdt ved romtemperatur i 13 timer. Det uløselige urea-biproduktet ble fjernet ved filtrering, 45 mg of methotrexate was dissolved in 1.0 ml of N,N-dimethylformamide and 25 mg of N-hydroxysuccinimide was added. 41 mg of N,N-dicyclohexyl carbodiimide was dissolved, and the mixture was kept at room temperature for 13 hours. The insoluble urea by-product was removed by filtration,
og 100 ul av filtratet ble satt til en løsning som inneholdt 5 mg humant serum albumin i 80 0 pl 0,1 M natriumfosfatbuffer ved pH 7,5 pluss 100 pl dioksan. and 100 µl of the filtrate was added to a solution containing 5 mg of human serum albumin in 800 µl of 0.1 M sodium phosphate buffer at pH 7.5 plus 100 µl of dioxane.
Denne reaksjonsblandingen ble holdt ved romtemperatur This reaction mixture was kept at room temperature
i 30 minutter, og satt på en "Sephadex G-25" kolonne som var ekvilibrert med 0,1 M natriumfosfatbuffer pH 7,5. Kolonnen ble utviklet med denne bufferen, og to eluerings-topper som inneholdt metotreksat ble erholdt, hvorav den første inneholdt metotreksat-humant serum albumin-konjugat. for 30 minutes, and applied to a "Sephadex G-25" column equilibrated with 0.1 M 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 methotrexate-human serum albumin conjugate.
Syntese av metotreksat- aminoetylmorfin- konjugat. Synthesis of methotrexate aminoethylmorphine conjugate.
a) Syntese av O 3-aminoetylmorfin. a) Synthesis of O 3-aminoethylmorphine.
I 10 ml tetrahydrofuran (THF) nettopp destillert over In 10 ml tetrahydrofuran (THF) just distilled over
litiumaluminiumhydrid (LAH) ble 400 mg LAH oppslemmet under nitrogen. En løsning av 400 mg morfin og 400 mg kloracetonitril i 4 ml friskt destillert THF ble tilsatt i løpet av 5 minutter, etterfulgt av tilbakeløpskoking i 1 time. Blandingen fikk kjøles, og 0,6 ml vann ble tilsatt etterfulgt av 0,6 ml 10 vekt% natriumhydroksyd og 2 ml vann. Etter filtrering av blandingen ble saltene vasket med THF, THF-fraksjonene slått sammen, tørket over magnesiumsulfat under nitrogen, filtrert, og filtratet inndampet, hvilket ga 380 mg O 3-aminoetylmorfin. lithium aluminum hydride (LAH), 400 mg of LAH was slurried under nitrogen. A solution of 400 mg morphine and 400 mg chloroacetonitrile in 4 mL freshly distilled THF was added over 5 min, followed by reflux for 1 h. The mixture was allowed to cool and 0.6 ml of water was added followed by 0.6 ml of 10% by weight sodium hydroxide and 2 ml of water. After filtering the mixture, the salts were washed with THF, the THF fractions combined, dried over magnesium sulfate under nitrogen, filtered, and the filtrate evaporated to give 380 mg of O 3 -aminoethylmorphine.
b) Syntese av N-t-butoksykarboksyl- jf - (O 3-aminoetylmorf in) - b) Synthesis of N-t-butoxycarboxyl-cf - (O 3-aminoethylmorphine) -
glutaminsyre--benzylester. glutamic acid--benzyl ester.
En blanding av O 3-aminoetylmorfin (50 mg fremstilt som ovenfor), N-t-BOC-glutaminsyre-cC-benzyl-ester (34 mg) A mixture of O 3-aminoethylmorphine (50 mg prepared as above), N-t-BOC-glutamic acid cC-benzyl ester (34 mg)
og dicykloheksylkarbodiimid (2 5 mg) i diklormetan (5 ml) ble rørt 4 timer ved romtemperatur. Blandingen ble fortynnet med etylacetat og vasket med fortynnet natriun-karbonatoppløsning. Etylacetatløsningen ble så ekstrahert to ganger med 0,1 N saltsyre, og de kombinerte sure ekstraktene ble behandlet med tilstrekkelig 10 vekt% natriunhydroksyd-løsning til å justere pH til 8,0. Løsningen ble ekstrahert to ganger med etylacetat, og 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 solution. The ethyl acetate solution was then extracted twice with 0.1 N hydrochloric acid, and the combined acid extracts were treated with sufficient 10% by weight sodium hydroxide solution to adjust the pH to 8.0. The solution was extracted twice with ethyl acetate, and
det kombinerte etylacetat ble vasket med koksaltløsning, tørket (tørt natriumfosfat) og inndampet til det ga en fargeløs olje (36 mg). c) Syntese av glutaminsyre- «C - (O 3-amidoetylmorf in) - cf - benzylester-trifluoreddiksyresalt. the combined ethyl acetate was washed with brine, dried (dry sodium phosphate) and evaporated to give a colorless oil (36 mg). c) Synthesis of glutamic acid- «C - (O 3-amidoethylmorphine) - cf - benzyl ester trifluoroacetic acid salt.
En løsning av N-t-BOC-glutaminsyre-morfinderivat (36 mg, fremstilt som ovenfor) i diklormetan (3 ml) ble rørt ved romtemperatur, og trifluoreddiksyre (1 ml) ble tilsatt. Blandingen ble rørt 15 minutter, og så inndampet til tørr-het. Resten var et fargeløst glass (40 mg). d) Syntese av metotreksat-$ -(0 3-aminoetylmorfin)- <£ - benzylester. A solution of N-t-BOC-glutamic acid morphine derivative (36 mg, prepared as 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 evaporated to dryness. The residue was a colorless glass (40 mg). d) Synthesis of methotrexate -(0 3-aminoethylmorphine)- <£ - benzyl ester.
En løsning av 4-amino-4-deoksy-N"'"^-metylpteronsyre (37 mg) i 3 ml dimetylsulfoksyd (DMSO) ble under røring ved romtemperatur behandlet med trietylamin (28 ul) og i-butyl-klorformat (25 ul), og blandingen ble rørt i 30 minutter. Den ble så satt til en blanding av morfin-derivatet (75 mg fremstilt som beskrevet under c) og trietylamin (28 ul) i DMSO (2 ml), og blandingen ble rørt ved 60°C i en time. Den avkjølte blandingen ble fortynnet med vann og ekstrahert to ganger med etylacetat. De kombinerte etylacetat-ekstrakter ble vasket med vann og så ekstrahert to ganger med 0,1 N saltsyre. De samlede sure ekstraktene ble behandlet med tilstrekkelig 10 vekt% natriumhydroksyd til å heve pH til 8,0. Blandingen ble ekstrahert tre ganger med etylacetat og A solution of 4-amino-4-deoxy-N"'"^-methylpteronic acid (37 mg) in 3 ml of dimethylsulfoxide (DMSO) was treated with triethylamine (28 µl) and i-butyl chloroformate (25 µl) while stirring at room temperature ), and the mixture was stirred for 30 min. It was then added to a mixture of the morphine derivative (75 mg prepared as described under c) and triethylamine (28 µl) in DMSO (2 ml) and the mixture was stirred at 60°C for one hour. The cooled mixture was diluted with water and extracted twice with ethyl acetate. The combined ethyl acetate extracts were washed with water and then extracted twice with 0.1 N hydrochloric acid. The pooled acid extracts were treated with sufficient 10% by weight sodium hydroxide to raise the pH to 8.0. The mixture was extracted three times with ethyl acetate and
de samlede ekstraktene vasket med koksaltløsning, tørket (tørt natriumsulfat) og inndampet, hvilket ga en gul olje (15 mg). Denne ble renset ved preparativ tynnsjikt-kromatografi (TLC) på kiselgel med systemet kloroform/ metanol, 4:1. Den ønskede forbindelsen erholdtes som et gult, fast stoff ( 4 mg). e) Syntese av metotreksat- jf - ( O 3-amidoetylmorf in) . the combined extracts were washed with brine, dried (dry sodium sulfate) and evaporated to give a yellow oil (15 mg). This was purified by preparative thin-layer chromatography (TLC) on silica gel with the system chloroform/methanol, 4:1. The desired compound was obtained as a yellow solid (4 mg). e) Synthesis of methotrexate - cf - ( O 3-amidoethylmorphine) .
Produktet fremstilt i d (4 mg) ble blandet med 0,1 N The product prepared in d (4 mg) was mixed with 0.1 N
natriumhydroksyd-løsning (5 ml), og blandingen ble rørt ved romtemperatur i 8 timer, hvilket resulterte i en klar gul løsning. sodium hydroxide solution (5 mL), and the mixture was stirred at room temperature for 8 h, resulting in a clear yellow solution.
Denne ble tilsatt o,1 N saltsyre (5 ml) og blandingen ble fylt opp til 25 ml med natriumortofosfat-buffer (0,05 M, pH 7,4), hvilket ga en løsning av den ønskede forbindelse som var egnet for bruk i enzymmålingen. To this was added 0.1 N hydrochloric acid (5 ml) and the mixture was made up to 25 ml with sodium orthophosphate buffer (0.05 M, pH 7.4), giving a solution of the desired compound suitable for use in the enzyme measurement.
Syntese av ferritin- metotreksat- konjugat. Synthesis of ferritin- methotrexate conjugate.
En løsning av humant leverferritin (1,3 mg) i natriumfosfatbuffer (0,05 M, pH 8,0, 5 ml) og 1 ml dimetyl-formamid (DMF) ble rørt ved romtemperatur, og 100 ul av en løsning oppnådd ved å behandle metotreksat (2 3 mg) i DMF (2 ml) med trietylamin (21 ul) og i-butylklorformat (15 ul) ved romtemperatur i 30 minutter ble tilsatt. Blandingen ble rørt ved romtemperatur i 2 timer, og så dialysert mot 2x2 liter natriumortofosfatbuffer (0,05 M, pH 7,4 inneholdende natriumklorid 0,1 M og natriumazid 0,05 vekt%) i 24 timer. Løsningen ble så sendt gjennom en kolonne av "Sephadex G-25" innstilt med den samme buffer, og de ferritinholdige fraksjoner ble slått sammen og fyllt opp til 10 ml med buffer. A solution of human liver ferritin (1.3 mg) in sodium phosphate buffer (0.05 M, pH 8.0, 5 ml) and 1 ml of dimethylformamide (DMF) was stirred at room temperature, and 100 μl of a solution obtained by treated methotrexate (2 3 mg) in DMF (2 ml) with triethylamine (21 µl) and i-butyl chloroformate (15 µl) at room temperature for 30 min was added. The mixture was stirred at room temperature for 2 hours, and then dialyzed against 2x2 liters of sodium orthophosphate buffer (0.05 M, pH 7.4 containing sodium chloride 0.1 M and sodium azide 0.05% by weight) for 24 hours. The solution was then passed through a column of "Sephadex G-25" adjusted with the same buffer, and the ferritin-containing fractions were pooled and made up to 10 ml with buffer.
Den resulterende løsning er egnet for bruk i en enzym-immuno-måling for bestemmelsen av ferritin. The resulting solution is suitable for use in an enzyme-immunoassay for the determination of ferritin.
De følgende eksempler illustrerer oppfinnelsen: The following examples illustrate the invention:
EKSEMPEL 1. EXAMPLE 1.
Enzyminhibitor- immunomåling for digoksin. Enzyme inhibitor immunoassay for digoxin.
100 ul ble inkubert ved 30°C i 15 minutter med 100 ul antidigoksin antistoffløsning, 100 pl NADPH-løsning, 100 µl was incubated at 30°C for 15 minutes with 100 µl antidigoxin antibody solution, 100 µl NADPH solution,
100 pl 2-merkaptoetanol-løsning og 550 pl natriumfosfat-buf fer pH 7,5. 100 pl metotreksat-digoksin-konjugat fra tilsvarende fremstillingseksempel i beskrivelsen (70 ug pr. ml) ble tilsatt, og blandingen ble inkubert i 15 minutter, hvorpå 100 pl dihydrofolatreduktase-oppløsning ble tilsatt. Dihydrofolatreduktase-fremstillingen som ble anvendt ble isolert fra kylling-lever ved metoden til Kaufman, B.T., & Gardiner, R.C., Journal of Biological Chemistry, Vol. 211, p. 1319 (1966). Blandingen ble inkubert ytterligere 3 minutter, og enzymaktiviteten bestemt ved tilsetning ~ av 100 ul dihydrofolatløsning og målt ved 340 nm i et variant spektrofotometer. Resultatene er vist i tabellen. 100 µl 2-mercaptoethanol solution and 550 µl sodium phosphate buffer pH 7.5. 100 µl of methotrexate-digoxin conjugate from the corresponding preparation example in the description (70 µg per ml) was added, and the mixture was incubated for 15 minutes, after which 100 µl of dihydrofolate reductase solution was added. The dihydrofolate reductase preparation used 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 an additional 3 minutes, and the enzyme activity determined by the addition of ~100 µl of dihydrofolate solution and measured at 340 nm in a variant spectrophotometer. The results are shown in the table.
Reaktantene ble tilsatt i den ovenfor beskrevne rekkefolge. OD = optisk densitet. The reactants were added in the order described above. OD = optical density.
Det fremgår av de ovenstående resultater at noen få ng digoksin i serum kan bestemmes i det beskrevne systemet. It appears from the above results that a few ng of digoxin in serum can be determined in the described system.
EKSEMPEL 2 EXAMPLE 2
Enzyminhibitor- immunomåling for humant serum albumin. Enzyme inhibitor immunoassay for human serum albumin.
100 ul fortynnet seum ble inkubert ved 30°C i 15 minutter med 100 ul antihuman serumalbumin-antistoff (kanin)-opp-løsning, 100 pl NADPH-oppløsning, 100 pl 2-merkaptoetanol-oppløsning og 550 pl natriumfosfatbuffer pH 7,5. 100 pl metotreksat/humant serumalbumin-konjugat (8 pg/ml) ble tilsatt, og blandingen inkubert i 15 minutter, hvoretter 100 ul dihydrofolatreduktase-oppløsning ble tilsatt. Blandingen ble inkubert i ytterligere 3 minutter, og enzymaktiviteten bestemt ved tilsetning av 100 pl dihydrofolat-oppløsning og målt ved 3 40 nm på et variant spektrofotometer. Resultatene er vist i tabell II. 100 µl of diluted serum was incubated at 30°C for 15 minutes with 100 µl of anti-human serum albumin antibody (rabbit) solution, 100 µl of NADPH solution, 100 µl of 2-mercaptoethanol solution and 550 µl of sodium phosphate buffer pH 7.5. 100 µl methotrexate/human serum albumin conjugate (8 µg/ml) was added and the mixture incubated for 15 minutes, after which 100 µl dihydrofolate reductase solution was added. The mixture was incubated for a further 3 minutes and the enzyme activity determined by adding 100 µl of dihydrofolate solution and measured at 3 40 nm on a variant spectrophotometer. The results are shown in Table II.
Det fremqår av de ovenstående resultater at noen få pg human serumalbumin kan bestemmes i det beskrevne systemet. It appears from the above results that a few pg of human serum albumin can be determined in the described system.
EKSEMPEL 3 EXAMPLE 3
Enzyminhibitor- immunomåling for morfin. Enzyme inhibitor immunoassay for morphine.
En blanding av 10 pl morfinløsning i passende konsentrasjon, 50 ul metotreksat -^-(0 3-amidoetylmorfin)-løsning (4 ng/ A mixture of 10 µl morphine solution at the appropriate concentration, 50 µl methotrexate -^-(0 3-amidoethylmorphine) solution (4 ng/
25 ml), 50 pl antimorfin-antistoffløsning, 10 pl NADPH-løsning, 10 pl 2-merkaptoetanol-løsning, 5C pl kalium-klorid-løsning, 150 pl tris-HCl-buffer (pH 7,5) inneholdende EDTA og 10 pl dihydrofolatreduktase (E.casei) løsning ble inkubert 2 0 minutter ved 3 7°C. Enzymaktiviteten i blandingen ble bestemt etter tilstetning av 10 pl dihydrofolat-løsning ved måling av forandringen i ekstink-sjon av løsningen ved 340 nm ved bruk av en "Centrifichem"-sentrifugalanalyse. Resultatene er vist i tabell III. 25 ml), 50 µl antimorphine antibody solution, 10 µl NADPH solution, 10 µl 2-mercaptoethanol solution, 5C µl potassium chloride solution, 150 µl tris-HCl buffer (pH 7.5) containing EDTA and 10 µl dihydrofolate reductase (E.casei) solution was incubated for 20 minutes at 37°C. The enzyme activity in the mixture was determined after addition of 10 µl of dihydrofolate solution by measuring the change in extinction of the solution at 340 nm using a "Centrifichem" centrifugal assay. The results are shown in Table III.
Den ovenstående tabell viser at med økende konsentrasjon av morfin avtar aktiviteten av dihydrofolatreduktase. Slike løsninger av morfin som inneholder 4 pg - 4 mg/ml morfin kunne bestemmes selv hvor bare 10 pl løsning var tilgjengelig. Dette betyr imidlertid ikke at dette er det krevede område, men snarere at det kan anvendes med hell. The above table shows that with increasing concentration of morphine, the activity of dihydrofolate reductase decreases. Such solutions of morphine containing 4 µg - 4 mg/ml morphine could be determined even where only 10 µl of solution was available. However, this does not mean that this is the required area, but rather that it can be used successfully.
Claims (1)
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GB10859/77A GB1595101A (en) | 1977-03-15 | 1977-03-15 | Enzyme modifier immunoassay |
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NO844764L NO844764L (en) | 1978-09-18 |
NO154814B true NO154814B (en) | 1986-09-15 |
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NO84844764A NO154814C (en) | 1977-03-15 | 1984-11-29 | PROCEDURE FOR IMMUNOLOGICAL DETERMINATION. |
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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) |
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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 |
NZ507994A (en) * | 1998-05-15 | 2003-01-31 | Sekisui Chemical Co Ltd | Immunoassay reagent 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 |
WO2008098789A2 (en) * | 2007-02-16 | 2008-08-21 | Ktb Tumorforschungsgesellschaft Mbh | Dual acting prodrugs |
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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 IL IL54234A patent/IL54234A0/en unknown
- 1978-03-08 AU AU33965/78A patent/AU519326B2/en not_active Expired
- 1978-03-14 BE BE185901A patent/BE864856A/en not_active IP Right Cessation
- 1978-03-14 FR FR7807271A patent/FR2384262A1/en active Granted
- 1978-03-14 AT AT0182178A patent/AT367203B/en not_active IP Right Cessation
- 1978-03-14 ES ES467831A patent/ES467831A1/en not_active Expired
- 1978-03-14 NO NO780902A patent/NO152955C/en unknown
- 1978-03-14 SE SE7802923A patent/SE447026B/en not_active IP Right Cessation
- 1978-03-14 DK DK114778A patent/DK152313C/en not_active IP Right Cessation
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- 1978-03-15 DE DE19782811257 patent/DE2811257A1/en not_active Ceased
- 1978-03-15 IT IT21256/78A patent/IT1158665B/en active
- 1978-12-01 ES ES475984A patent/ES475984A1/en not_active Expired
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1984
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ATA182178A (en) | 1981-10-15 |
DK152313B (en) | 1988-02-15 |
AU519326B2 (en) | 1981-11-26 |
IT7821256A0 (en) | 1978-03-15 |
IL54234A0 (en) | 1978-06-15 |
DE2811257A1 (en) | 1978-09-21 |
NO152955C (en) | 1985-12-18 |
AT367203B (en) | 1982-06-11 |
AU3396578A (en) | 1979-09-13 |
FR2384262A1 (en) | 1978-10-13 |
NO152955B (en) | 1985-09-09 |
NO780902L (en) | 1978-09-18 |
BE864856A (en) | 1978-09-14 |
NO844764L (en) | 1978-09-18 |
CA1102789A (en) | 1981-06-09 |
IT1158665B (en) | 1987-02-25 |
DK114778A (en) | 1978-09-16 |
DK152313C (en) | 1988-09-26 |
NL7802845A (en) | 1978-09-19 |
SE447026B (en) | 1986-10-20 |
GB1595101A (en) | 1981-08-05 |
ES475984A1 (en) | 1979-06-16 |
ES467831A1 (en) | 1979-09-01 |
SE7802923L (en) | 1978-09-16 |
FR2384262B1 (en) | 1983-04-08 |
JPS53115814A (en) | 1978-10-09 |
CH641570A5 (en) | 1984-02-29 |
NO154814C (en) | 1986-12-29 |
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