NO159620B - IMMUNOLOGICAL DETERMINATION METHOD. - Google Patents
IMMUNOLOGICAL DETERMINATION METHOD. Download PDFInfo
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- NO159620B NO159620B NO811407A NO811407A NO159620B NO 159620 B NO159620 B NO 159620B NO 811407 A NO811407 A NO 811407A NO 811407 A NO811407 A NO 811407A NO 159620 B NO159620 B NO 159620B
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- cea
- substance
- immunologically active
- active reaction
- reaction partner
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
Description
Foreliggende oppfinnelse vedrører en enzymatisk immunologisk metode ifølge det såkalte "sandwich-prinsipp". Ifølge dette prinsippet bringes substansen som skal bestemmes,hvilken kan være et antigen, et antistoff eller et hapten til omsetning med to immunologisk aktive reaksjonspartnere med etterfølgende adskillelse som angitt i krav l's ingress. The present invention relates to an enzymatic immunological method according to the so-called "sandwich principle". According to this principle, the substance to be determined, which can be an antigen, an antibody or a hapten, is brought into contact with two immunologically active reaction partners with subsequent separation as stated in the preamble of claim 1.
Som regel er en av disse immunologiske aktive reaks jonspartnere bundet til en vannuløselig bærer, mens den andre er forsynt med et egnet enzym. I praksis bringes substansen som skal bestemmes først i en omsetning med den reaksjonspartner som As a rule, one of these immunologically active reaction partners is bound to a water-insoluble carrier, while the other is provided with a suitable enzyme. In practice, the substance to be determined is first brought into circulation with the reaction partner which
er bundet til en bærer, hvorpå, etter faseskille og vask den substans som i mellomtiden er immunologisk bundet til bæreren bringes til omsetning med den andre, enzymmerkete reaksjonspartner. is bound to a carrier, after which, after phase separation and washing, the substance which is in the meantime immunologically bound to the carrier is brought into contact with the other, enzyme-labelled reaction partner.
Etter nytt faseskille skjer den enzymatiske reaksjon for kvantitativ påvisning med substansen som skal bestemmes enten After a new phase separation, the enzymatic reaction for quantitative detection takes place with the substance to be determined either
i den faste eller flytende fase. in the solid or liquid phase.
I DE-OS 27 44 836 blir det beskrevet en immunokjemisk målfremgangsmåte som angår en kombinasjon av sandwich-metoden og den konkurrerende metode og som er særpreget ved at etter omsetning mellom antigenene som skal måles og de uløseliggjorte antistoff blir de merkete antistoff bragt til antigenet av antigen/antistoff-komplekset uten fjerning av reaksjonsblandingen for kontinuerlig omsetning. DE-OS 27 44 836 describes an immunochemical target method which relates to a combination of the sandwich method and the competitive method and which is characterized by the fact that after reaction between the antigens to be measured and the insoluble antibodies, the labeled antibodies are brought to the antigen by the antigen/antibody complex without removing the reaction mixture for continuous reaction.
I DE-OS 29 25 565 blir det beskrevet en entrinns-sandwich- DE-OS 29 25 565 describes a single-stage sandwich
måte som blir eksemplifisert ved en radioimmunofremgangsmåte under anvendelse av polyklonale antistoff. Anvendelsen av monoklonale antistoff henh. anvendelse av et monoklonalt antistoff og et polyklonalt antistoff såvel som anvendelsen av en enzymimmunofremgangsmåte blir ved denne publikasjon på ingen måte nærliggende. manner exemplified by a radioimmuno-method using polyclonal antibodies. The use of monoclonal antibodies acc. the use of a monoclonal antibody and a polyclonal antibody as well as the use of an enzyme immuno-method are in no way apparent in this publication.
Innenfor rammen av foreliggende oppfinnelse fant man nå over-raskende at man i motsetning til den tidligere oppfatning kan arbeide i en "entrinns-metode" ved hvilken substansen som skal bestemmes samtidig bringes til omsetrting med de to immunologisk aktive reaksjonspartnere i en enkel inkubasjon, og den kan også utføres når man som immunologisk reaksjonspartner setter inn to forskjellige monoklonale antistoff og et antistoff fra en forskjellig dyrefamilie. Within the framework of the present invention, it was now surprisingly found that, contrary to the previous opinion, one can work in an "entry-step method" in which the substance to be determined is simultaneously brought into circulation with the two immunologically active reaction partners in a simple incubation, and it can also be carried out when two different monoclonal antibodies and an antibody from a different animal family are used as immunological reaction partners.
Foreliggende oppfinnelse vedrører følgelig en enzymatisk immunologisk fremgangsmåte for påvisning hhv. bestemmelse av en substans med en immunologisk aktiv reaksjonspartner, hvilken er utstyrt med et enzym, samt en immunologisk aktiv reaksjonspartner som er eller blir bundet til en vannuløslig bærer, gjennom inkubering av substansen som skal bestemmes The present invention therefore relates to an enzymatic immunological method for detecting or determination of a substance with an immunologically active reaction partner, which is equipped with an enzyme, as well as an immunologically active reaction partner that is or is bound to a water-insoluble carrier, through incubation of the substance to be determined
med de immunologisk aktive reaksjonspartnere, etterfølgende adskillelse av fast og flytende fase og måling av graden av enzym-merkLng enten i den faste eller flytende fase som with the immunologically active reaction partners, subsequent separation of solid and liquid phase and measurement of the degree of enzyme labeling either in the solid or liquid phase which
mål for den mengde av substans som skal bestemmes, hvilken measure of the amount of substance to be determined, which
er særpreget ved de trekk som er angitt i krav l's karakteriserende del . is characterized by the features specified in claim l's characterizing part.
Som substans som skal bestemmes kan det nevnes alle bestanddeler i fysiologiske væsker, celle- eller vevsekstrakter, for hvilke immunologiske reaksjonspartnere eksisterer eller kan dannes, og som har to eller flere immunologisk aktive punkter. Til disse hører peptider, proteiner, lipoproteiner, glykoproteiner, steroler, steroider, lipoider/ nukleinsyrer, enzymer, hormoner, polysaccarider, alkaloider. Foretrukne immunologisk aktive substanser er de naturlige antigener så som hormoner, enzymer, organspesifikke antigener, bindevevs-komponenter, blodcelleanti-gener, plasmaproteiner, patologiske globuliner. Særlig foretrukne substanser er det carcinoembryo-nale antigen (CEA) samt det humane coriogonadotropin (HCG). All components in physiological fluids, cell or tissue extracts, for which immunological reaction partners exist or can be formed, and which have two or more immunologically active points can be mentioned as substances to be determined. These include peptides, proteins, lipoproteins, glycoproteins, sterols, steroids, lipoids/nucleic acids, enzymes, hormones, polysaccharides, alkaloids. Preferred immunologically active substances are the natural antigens such as hormones, enzymes, organ-specific antigens, connective tissue components, blood cell antigens, plasma proteins, pathological globulins. Particularly preferred substances are the carcinoembryonic antigen (CEA) and the human choriogonadotropin (HCG).
Ved metoden ifølge oppfinnelsen tjener den immunologisk aktive reaksjonspartner, som er forsynt med et enzym, som indikator for den immunologiske reaksjon. Foretrukne enzymer er alkal-isk fosfatase, malat-dehydrogenase, glukose-6-fosfat-dehydrogenase, glucose-oxidase, glucoamylase, galaktosidase og ace-tylcolinesterase. Et særlig foretrukket enzym er peroxidase fra pepperrot. In the method according to the invention, the immunologically active reaction partner, which is provided with an enzyme, serves as an indicator for the immunological reaction. Preferred enzymes are alkaline phosphatase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase, galactosidase and acetylcholinesterase. A particularly preferred enzyme is horseradish peroxidase.
Enzymet som indikator for den immunologiske reaksjon måles ifølge kjente metoder i den flytende, eller fortrinnsvis i den faste fase, og er et mål for den mengde av substans som skal bestemmes. Ved peroxidasen fra pepperrot som markør, måles fortrinnsvis enzymmengden på grunnlag av den tilstede-værende katalytiske aktivitet, hvilken bestemmes ved hjelp av ^ 2^ 2 °^ o-fenylendiamin som redoxindikator. Derunder måles etter en 30 minutters katalytisk reaksjon fargeintensiteten til den oksyderte redoxindikator fotometrisk. The enzyme as an indicator of the immunological reaction is measured according to known methods in the liquid, or preferably in the solid phase, and is a measure of the amount of substance to be determined. With the peroxidase from horseradish as a marker, the amount of enzyme is preferably measured on the basis of the catalytic activity present, which is determined using ^ 2^ 2 °^ o-phenylenediamine as a redox indicator. Underneath, after a 30-minute catalytic reaction, the color intensity of the oxidized redox indicator is measured photometrically.
Den andre immunologisk aktive reaksjonspartner tjener til adskillelsen av substansen som skal bestemmes fra analyse-prøven. For dette adskillelsestrinn kan den andre immunologisk aktive reaksjonspartneren fra begynnelsen av være bundet til en vannuløselig bærer eller kan under eller etter den immunologiske reaksjonen bindes til en tilsvarende bærer. The second immunologically active reaction partner serves for the separation of the substance to be determined from the analysis sample. For this separation step, the second immunologically active reaction partner can be bound to a water-insoluble carrier from the beginning or can be bound to a corresponding carrier during or after the immunological reaction.
Som vannuløselig bærer for den andre immunologisk aktive reaksjonspartner er følgende egnet: organiske og uorganiske polymere (amylase, dextraner, opprinnelig eller modifisert cellulose, polyacrylamid, agarose, magnetitt,porøst glass-pulver, polyvinylidenfluorid (Latex), innvendig vegg av forsøksbeholdere (prøverør, titreringsplater eller kyvetter av glass eller kunststoff1 samt overflaten av faste legemer (glass- og plaststav, stav med fortykkelse på enden, stav med vinger eller lameller på enden). Særlig egnede bær-ere for metoden ifølge oppfinnelsen er glass- og plastkuler. As a water-insoluble carrier for the second immunologically active reaction partner, the following are suitable: organic and inorganic polymers (amylase, dextrans, original or modified cellulose, polyacrylamide, agarose, magnetite, porous glass powder, polyvinylidene fluoride (Latex), inner wall of test containers (test tubes, titration plates or cuvettes made of glass or plastic1 as well as the surface of solid bodies (glass and plastic rod, rod with thickening at the end, rod with wings or lamellae at the end). Particularly suitable carriers for the method according to the invention are glass and plastic balls.
Den andre immunologisk aktive reaksjonspartner kan være fysikalsk eller kjemisk bundet til den vannuløslige bærer (adsorptiv^eller kan, ved1 hjelp av en ytterligere reaks jonspart-ner som på sin side igjen er bundet til en bærer, bindes under eller etter reaksjonen. The other immunologically active reaction partner can be physically or chemically bound to the water-insoluble carrier (adsorptive) or can, with the help of a further reaction partner which is in turn bound to a carrier, be bound during or after the reaction.
Vesentlig for metoden ifølge oppfinnelsen er at substansen Essential to the method according to the invention is that the substance
som skal bestemmes har minst to immunologisk aktive punkter (epitoper), hvilke kan gjenkjennes av de to immunologisk aktive reaksjonspartnere, den reaksjonspartner som er bundet til en bærer og den reaksjonspartner som er utstyrt med et enzym, to be determined has at least two immunologically active points (epitopes), which can be recognized by the two immunologically active reaction partners, the reaction partner bound to a carrier and the reaction partner equipped with an enzyme,
og kan bringes til reaksjon. Som immunologisk aktive reaksjonspartnere benyttes fortrinnsvis to forskjellige komponenter som riktignok begge reagerer med substansen som skal bestemmes, men hver er rettet mot forskjellige immunologisk aktive punkter. For bestemmelse av antigener er spesielt to antistoffer for dette antigenet egnet, hvilke produseres i to forskjellige dyrearter og som er rettet mot forskjellige epitoper av dette antigenet. Særlig egnet er kombinasjonen av antistoffer av forskjellige kloner eller av monoklonale antistoffer og antist°ffer fra en forskjellig dyreart. and can be brought to reaction. As immunologically active reaction partners, two different components are preferably used which both react with the substance to be determined, but each is directed at different immunologically active points. For the determination of antigens, two antibodies for this antigen are particularly suitable, which are produced in two different animal species and which are directed against different epitopes of this antigen. Particularly suitable is the combination of antibodies of different clones or of monoclonal antibodies and antibodies from a different animal species.
For den immunologiske reaksjon kan prøven med substansen for bestemmelse bli tilsatt direkte eller fortynnet på forhånd, eller være forbehandlet på egnet måte. Til forbehandling kan substansen til bestemmelse bli isolert, anriket eller frigjort for forstyrrende bestanddeler. For the immunological reaction, the sample with the substance for determination can be added directly or diluted in advance, or be pre-treated in a suitable way. For pretreatment, the substance to be determined can be isolated, enriched or freed from interfering components.
Ifølge fremgangsmåten i henhold til oppfinnelsen blir substansen til bestemmelse samtidig bragt til omsetning med den enzym-merkete såvel som den bærerbundete immunologisk aktive reaksjonspartner. Rekkefølgen til reagenstilsetningen er avhengig av arten til det valgte bærersystem. Under arbeidet med en sensibilisert plastik-kule blandes først den enzym-merkete reaksjonspartner sammen med substansen som skal bestemmes i et egnet prøverør og tilsettes deretter kulen som er sensibilisert med den andre reaksjonspartner. According to the method according to the invention, the substance to be determined is simultaneously reacted with the enzyme-labeled as well as the carrier-bound immunologically active reaction partner. The order of reagent addition depends on the nature of the carrier system chosen. When working with a sensitized plastic ball, the enzyme-labeled reaction partner is first mixed with the substance to be determined in a suitable test tube and then added to the ball that has been sensitized with the other reaction partner.
Den immunologiske reaksjonen utføres under bibehold av en optimal pH-verdi som kan ligge mellom 4 og 9 i et egnet puf-fersystem. Foretrukne puffere er f.eks. acetatpuffer, citrat-puffer, fosfatpuffer, tris-puffer, trietanolaminpuffer, borat-puffer eller glycinpuffer. Det kan også anvendes puffer-blandinger. The immunological reaction is carried out while maintaining an optimal pH value which can lie between 4 and 9 in a suitable buffer system. Preferred puffers are e.g. acetate buffer, citrate buffer, phosphate buffer, tris buffer, triethanolamine buffer, borate buffer or glycine buffer. Puffer mixtures can also be used.
Den immunologiske reaksjonen skjer fortrinnsvis ved en tempe-ratur mellom 0 og 55°C. Normalt tiltar den immunologiske reak-sjonshastighet med høyere temperaturer, hvor ved like- The immunological reaction preferably takes place at a temperature between 0 and 55°C. Normally, the immunological reaction rate increases with higher temperatures, where at equal
vekten raskere oppnås under ellers like forsøksbetingelser. the weight is achieved more quickly under otherwise identical test conditions.
Inkubasjonen av substansen som skal bestemmes med den enzym-merkete reaksjonspartner og den bærer-bundne reaksjonspartner kan finne sted inntil likevekt oppnås. Den immunologiske reaksjonen kan imidlertid også avbrytes på et tidligere tids-punkt, idet den faste og flytende fase adskilles etter en bestemt inkubasjonsvarighet og graden av enzym-merking bestemmes enten i den flytende eller i den faste fase. The incubation of the substance to be determined with the enzyme-labeled reaction partner and the carrier-bound reaction partner can take place until equilibrium is reached. However, the immunological reaction can also be interrupted at an earlier point in time, as the solid and liquid phase are separated after a specific incubation period and the degree of enzyme labeling is determined either in the liquid or in the solid phase.
Ved den immunologiske reaksjon kan det treffes tiltak for å stabilisere den immunologiske aktiviteten til reaksjonspart-nerne og substansen som skal bestemmes samt enzymet. Videre kan inkubasjonsløsningen tilsettes bestanddeler så som proteiner og detergenter for å utelukke uspesifikke reaksjon-er, for å redusere hemmende påvirkning eller for-aktivering. During the immunological reaction, measures can be taken to stabilize the immunological activity of the reaction partners and the substance to be determined as well as the enzyme. Furthermore, components such as proteins and detergents can be added to the incubation solution to exclude non-specific reactions, to reduce inhibitory effects or pre-activation.
Den nye metoden ifølge foreliggende oppfinnelse er overor-dentlig ømfindtlig og utmerker seg ved at den er enkel å utføre. The new method according to the present invention is extremely sensitive and is distinguished by the fact that it is simple to perform.
De etterfølgende eksempler belyser oppfinnelsen. The following examples illustrate the invention.
EKSEMPLER EXAMPLES
Eksempel 1 Example 1
Kvantitativ bestemmelse av CEA i pasientplasmaer med et monoklonalt CEA- antistoff og et vanlig CEA antistoff ( geit) Quantitative determination of CEA in patient plasmas with a monoclonal CEA antibody and a normal CEA antibody (goat)
I de nødvendige antall prøverør (10 x 75 mm) pipeteres i hvert 0,2 mm prøveløsning (0,2 mol NaH2P04/Na2HPO^, pH 6,5 med 2 g pr. 1 storfeserumalbumin, 20% normalt geiteserum og 0,2 ug/ml geit-anti-CEA-peroxidase-konjugat), 0,050 ml av pasientplasma som skal analyseres hhv. CEA standard (0 ng/ml CEA 2,5 ng/ml CEA, 10 ng/ml og 20 ng/ml CEA) og CEA-kontrollserumet (5,0 ng/ml CEA - 1,0 ng/ml) tilblandes, hvert tilsettes en polystyrenkule (diam.=6,5 mm) sensibilisert med monoklonalt muse anti-CEA og inkuberes ved 3 7°C i 16 timer. Deretter vaskes polystyrenkulene 3 ganger med hver gang 2-5 ml dest. vann, over-føres i 0,5 ml substratpuffer for aktivitetsbestemmelse av peroxidase (0,1 mol/l kaliumcitratpuffer med pH 5,0 med 6 mmol pr. 1 H202 og 20 mmol/1 o-fenylendiamin) og inkuberes 30 min. ved romtemperatur (22°C). For avbrudd av peroksydaseaktivi-teten samt for intensivering av fargeintensiteten tilblandes 2,0 ml IN HC1 og i løpet av 30 min. måles ekstinksjonen ved bølgelengden 4 92 nm fotometrisk målt. I tabell 1 er verdiene for en CEA-bestemmelse oppført og sammenlignet med de verdier man får med radioimmunomålingen til Roche. In the required number of test tubes (10 x 75 mm), each 0.2 mm sample solution (0.2 mol NaH2P04/Na2HPO^, pH 6.5 with 2 g per 1 bovine serum albumin, 20% normal goat serum and 0.2 ug /ml goat anti-CEA peroxidase conjugate), 0.050 ml of patient plasma to be analyzed or The CEA standard (0 ng/ml CEA 2.5 ng/ml CEA, 10 ng/ml and 20 ng/ml CEA) and the CEA control serum (5.0 ng/ml CEA - 1.0 ng/ml) are mixed, each a polystyrene ball (diam.=6.5 mm) sensitized with monoclonal mouse anti-CEA is added and incubated at 37°C for 16 hours. The polystyrene balls are then washed 3 times with each time 2-5 ml dist. water, transferred into 0.5 ml substrate buffer for determination of activity of peroxidase (0.1 mol/l potassium citrate buffer with pH 5.0 with 6 mmol per 1 H202 and 20 mmol/1 o-phenylenediamine) and incubated for 30 min. at room temperature (22°C). To interrupt the peroxidase activity and to intensify the color intensity, 2.0 ml IN HC1 is mixed and within 30 min. the extinction is measured photometrically at the wavelength 4 92 nm. In table 1, the values for a CEA determination are listed and compared with the values obtained with the Roche radioimmunoassay.
x Fremstillingen av monoklonalt muse anti-CEA skjer analogt med den metode som er beskrevet i Journal of Immunological Methods, 32 (1980) 297-304 hvorunder myelomlinjen Sp 2/01-AG anvendes som utgangscellelinje for fusjonen, hvilken er depo-nert under nr. CRL 800 6 hos ATCC. Fusjonen skjer med milt-celler fra mus som er immunisert med CEA. Immuniseringen av musene skjedde analogt til tabell 1 i den nevnte publikasjon, hvorunder de første to immuniseringer skjedde begge med 50 ug CEA, immuniseringene 3 og 4 ble utelatt, immunisering 5 med x The production of monoclonal mouse anti-CEA takes place analogously to the method described in Journal of Immunological Methods, 32 (1980) 297-304, whereby the myeloma line Sp 2/01-AG is used as the starting cell line for the fusion, which is deposited under no. .CRL 800 6 at ATCC. The fusion takes place with spleen cells from mice immunized with CEA. The immunization of the mice took place analogously to table 1 in the aforementioned publication, during which the first two immunizations both took place with 50 ug CEA, immunizations 3 and 4 were omitted, immunization 5 with
50 ug CEA og immuniseringene 6-8 alle med 200 ug CEA. 50 ug CEA and immunizations 6-8 all with 200 ug CEA.
Verdier under 2,5 ng/ml CEA ligger i normalområdet, mens verdier over 2,5 ng/ml ligger i det patologiske området. Values below 2.5 ng/ml CEA are in the normal range, while values above 2.5 ng/ml are in the pathological range.
Eksempel 2 Example 2
Kvantitativ bestemmelse av CEA i pasientplasmaer med monoklonale CEA antistoffer fra to forskjellige kloner. Quantitative determination of CEA in patient plasmas with monoclonal CEA antibodies from two different clones.
I det nødvendige antall prøverør (10 x 75 mm) pipeteres i hvert 0,2 mm prøveløsning (0,2 mol/l NaH2P04/Na2HP04, pH In the required number of test tubes (10 x 75 mm) each 0.2 mm of sample solution (0.2 mol/l NaH2P04/Na2HP04, pH
6,5 med 2 g/l storfeserumalbumin, 20% normalt geiteserum og 0,15 ug/l monoklonalt mus-anti-CEA-peroxidase-konjugat ), 0,050 ml av pasientplasmaene som skal analyseres hhv. CEA-standardene (0 ng/ml CEA, 2,5 ng/ml CEA, 10 ng/ml CEA og 2 0 ng/ml CEA) og av CEA kontrollserumet (5,0 ng/ml CEA - 1,0 ng/ml) tilblandet, hvert tilsettes en polystyrenkule (diam.= 6,5 mm) sensibilisert med monoklonalt mus-anti-CEA og inkuberes 16 t ved 37°C. Deretter vaskes polystyrenkulene 3 ganger med 2-5 ml dest. vann, overføres i 0,5 ml substratpuffer for aktivitets-bestemmelsen av peroxidase (0,1 mol/l kaliumcitratpuffer med pH 5,0 med 6 mmol/1 H202 og 20 mmol/1 o-fenylendiamin) og inkuberes 3 0 min. ved romtemperatur (22 C). For å avbryte peroxidaseaktiviteten samt for intensivering av fargeintensiteten tilblandes 2,0 ml IN HC1 og i løpet av 3 0 min. måles ekstinksjonen ved bølgelengden 4 92 nm fotometrisk.I tabell III er verdien for en CEA-bestemmelse oppført og sammenlignet med de verdier som erholdtes med radioimmunmålingen fra Roche. 6.5 with 2 g/l bovine serum albumin, 20% normal goat serum and 0.15 ug/l monoclonal mouse anti-CEA peroxidase conjugate ), 0.050 ml of the patient plasmas to be analyzed respectively. the CEA standards (0 ng/ml CEA, 2.5 ng/ml CEA, 10 ng/ml CEA and 20 ng/ml CEA) and of the CEA control serum (5.0 ng/ml CEA - 1.0 ng/ml ) mixed, a polystyrene sphere (diam.= 6.5 mm) sensitized with monoclonal mouse anti-CEA is added to each and incubated for 16 h at 37°C. The polystyrene balls are then washed 3 times with 2-5 ml dist. water, transferred into 0.5 ml substrate buffer for the activity determination of peroxidase (0.1 mol/l potassium citrate buffer with pH 5.0 with 6 mmol/1 H202 and 20 mmol/1 o-phenylenediamine) and incubated for 30 min. at room temperature (22 C). To interrupt the peroxidase activity and to intensify the color intensity, 2.0 ml IN HC1 is mixed and during 30 min. the extinction at the wavelength 4 92 nm is measured photometrically. In table III the value for a CEA determination is listed and compared with the values obtained with the radioimmunoassay from Roche.
x x
Fremstillingen av monoklonalt mus anti-CEA skjer analogt etter den metode som er beskrevet i Journal of Immunological The production of monoclonal mouse anti-CEA occurs analogously to the method described in the Journal of Immunological
Méthods, 32 (1980) 297-304, hvorunder myelomlinjen Sp 2/01-Ag anvendes som utgangscellelinje for fusjonen, hvilken er depo-nert under nr. CRL 8006 hos ATCC. Fusjonen skjer med milt-celler av mus som er immunisert med CEA. Immuniseringen av musene fant sted analogt med tabell 1 i den nevnte publikasjon, idet de første to immuniseringer fant sted med 50 ug CEA, immuniseringene 3 og 4 ble utelatt, immunisering 5 med 50 jug CEA Méthods, 32 (1980) 297-304, whereby the myeloma line Sp 2/01-Ag is used as the starting cell line for the fusion, which is deposited under No. CRL 8006 at ATCC. The fusion takes place with spleen cells of mice immunized with CEA. The immunization of the mice took place analogously to table 1 in the aforementioned publication, the first two immunizations taking place with 50 µg CEA, immunizations 3 and 4 were omitted, immunization 5 with 50 µg CEA
og immuniseringene 6-8 med hver 200 rø CEA- Det anvendes to forskjellige egnede monoklonale antistoffer som er rettet mot forskjellige epitoper av CEA antigener. and the immunizations 6-8 with each 200 r CEA- Two different suitable monoclonal antibodies are used which are directed against different epitopes of CEA antigens.
Verdier under 2,5 ng/ml CEA ligger i normalområdet, mens verdier over 2,5 ng/ml ligger i det patologiske området. Values below 2.5 ng/ml CEA are in the normal range, while values above 2.5 ng/ml are in the pathological range.
Eksempel 3 Example 3
Kvantitativ bestemmelse av HCG 1 serum/ plasma/ urin: Quantitative determination of HCG 1 serum/ plasma/ urine:
1 det nødvendige antall prøverør (10 x 75 mm) pipeteres 0,2 ml prøveløsning (0,1 mol/l NaH2P04/Na2HP04, pH 7,0 med 2 g/l storfeserumalbumin, og 1,0 ug/ml monoklonal mus-anti-HCG-peroxidase-konjugat<x>), 0,050 ml av pasientplasmaene som skal analyseres hhv. HCG-standardene (0, 25, 50, 100 og 250 mIU/ml HCG) tilblandes, hvert tilsettes en polystyrenkule (diam.= 6,5 mm) sensibilisert med kanin-anti-HCG og inkuberes ved romtemperatur i 16 t i vannmettet atmosfære. Deretter vaskes polystyrenkulene tre ganger med 2--5 ml dest. vann, overføres i 0,5 ml substratpuffer for aktivitetsbestem-melsen av peroxidase (0,1 mol/l kaliumcitratpuffer med pH 5,0 med 6 mmol/1 H202 og 20 mmol/1 o-fenylendiamin) og inkuberes i 30 min. under romtemperatur (16*-30°C) . For å avbryte peroxidaseaktiviteten samt for intensivering av fargeintensiteten tilblandes 2,0 ml IN HC1 og ekstinksjonen måles fotometrisk i løpet av 30 min. med bølgelengden 492 nm. I tabellen er det oppført verdiene av en HCG-bestemmelse i serum og i urin. Into the required number of test tubes (10 x 75 mm), pipette 0.2 ml of sample solution (0.1 mol/l NaH2PO4/Na2HP04, pH 7.0 with 2 g/l bovine serum albumin, and 1.0 ug/ml monoclonal mouse anti -HCG-peroxidase conjugate<x>), 0.050 ml of the patient plasmas to be analyzed respectively. The HCG standards (0, 25, 50, 100 and 250 mIU/ml HCG) are mixed, a polystyrene sphere (diam.= 6.5 mm) sensitized with rabbit anti-HCG is added to each and incubated at room temperature for 16 h in a water-saturated atmosphere. The polystyrene balls are then washed three times with 2--5 ml dist. water, transferred into 0.5 ml substrate buffer for the activity determination of peroxidase (0.1 mol/l potassium citrate buffer with pH 5.0 with 6 mmol/1 H202 and 20 mmol/1 o-phenylenediamine) and incubated for 30 min. below room temperature (16*-30°C) . To interrupt the peroxidase activity and to intensify the color intensity, 2.0 ml of IN HC1 is mixed and the extinction is measured photometrically within 30 min. with the wavelength 492 nm. The table lists the values of an HCG determination in serum and in urine.
x x
Fremstilling av monoklonalt anti~-HCG kan skje etter en metode som er beskrevet i Journal of Immunological Methods 32 (1980) 297-304. Production of monoclonal anti~-HCG can take place according to a method described in Journal of Immunological Methods 32 (1980) 297-304.
HCG bestemmelse i serum og 1 urin. HCG determination in serum and 1 urine.
1. Standardkurver 1. Standard curves
2. Pasientprøver 2. Patient samples
Omregning: 1 ng rent HCG tilsvarer ca. 10 mlU HCG. Conversion: 1 ng of pure HCG corresponds to approx. 10 mlU HCG.
Eksempel 4 Example 4
Kvantitativ bestemmelse av HCG i serum med monoklonale HCG- antistoffer fra to forskjellige kloner. Quantitative determination of HCG in serum with monoclonal HCG antibodies from two different clones.
I det nødvendige antall prøverør (10 x 75 mm) pipeteres Pipette into the required number of test tubes (10 x 75 mm).
0,2 ml prøveløsning (0,1 mol/l NaH2P04/Na2HP04,pH 7,0 0.2 ml sample solution (0.1 mol/l NaH2P04/Na2HP04, pH 7.0
med 2 g/l storfeserumalbumin og 1,0 ug/ml monoklonalt mus-anti-HCG-peroxidase-konjugat jf),0,050 ml av pasientplasmaene som skal analyseres hhv. HCG-standardene (0, 25, 50, 100 og 200 mIU/ml HCG) tilblandes, hver tilføres en polystyrenkule ) (diam. = 6,5.mm) sensibilisert med monoklonalt mus-anti-HCG og inkuberes f.eks. i 2 timer ved 3 7°C. Deretter vaskes polystyrenkulene tre ganger med hver 2-5 ml dest. vann, overføres i 0,5 ml substratpuffer for aktivitetsbestemmelse av peroxidase (0,1 mol/l kaliumcitratpuffer med pH 5,0 med 6 mmol/1 H202 og 20 mmol/1 o^fenylendiamin) with 2 g/l bovine serum albumin and 1.0 ug/ml monoclonal mouse anti-HCG peroxidase conjugate cf), 0.050 ml of the patient plasmas to be analyzed respectively. The HCG standards (0, 25, 50, 100 and 200 mIU/ml HCG) are mixed, each added to a polystyrene ball ) (diam. = 6.5 mm) sensitized with monoclonal mouse anti-HCG and incubated e.g. for 2 hours at 37°C. The polystyrene beads are then washed three times with each 2-5 ml of distilled water. water, transferred into 0.5 ml substrate buffer for peroxidase activity determination (0.1 mol/l potassium citrate buffer with pH 5.0 with 6 mmol/1 H202 and 20 mmol/1 o^phenylenediamine)
og inkuberes i 30 min. ved romtemperatur (16-30 C). For å avbryte peroxidaseaktiviteten samt for intensivering av fargeintensiteten tilblandes 1,0 ml 2N HC1 og ekstinksjonen måles fotometrisk ved bølgelengden 492 nm i løpet av 30 min. I tabellen er verdiene for HCG-bestemmelsene i serum opp-ført. and incubated for 30 min. at room temperature (16-30 C). To interrupt the peroxidase activity and to intensify the color intensity, 1.0 ml of 2N HC1 is added and the extinction is measured photometrically at a wavelength of 492 nm during 30 min. In the table, the values for the HCG determinations in serum are listed.
Fremstillingen av monoklonale mus-HCG-antistoffer skjer ifølge den metode som er beskrevet i Journal of Immunological Meitods, 32 (1980) 297-304. Det anvendes to forskjellige egnede monoklonale antistoffer som er rettet mot forskjellige epitoper av HCG-antigenet. The production of mouse monoclonal HCG antibodies is carried out according to the method described in Journal of Immunological Methods, 32 (1980) 297-304. Two different suitable monoclonal antibodies are used which are directed against different epitopes of the HCG antigen.
HCG bestemmelse i humanserum (Middelverdi fra dobbel-bestemmeIsene) HCG determination in human serum (Mean value from double-determination)
j^ Standardkurver j^ Standard curves
HCG-verdiene til de nedenfor anførte eksempler fra pasientsera ble avlest fra standardkurven 1). Standardkurvene 2), 3) og 4) ble målt på en annen dag med nye HCG standarder. The hCG values of the examples listed below from patient sera were read from the standard curve 1). The standard curves 2), 3) and 4) were measured on another day with new HCG standards.
2. Pasientsera 2. Patient sera
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| EP0044219A1 (en) * | 1980-07-16 | 1982-01-20 | Unilever Plc | Methods of immuno analysis using monoclonal antibodies |
| US4486530A (en) * | 1980-08-04 | 1984-12-04 | Hybritech Incorporated | Immunometric assays using monoclonal antibodies |
| US4879219A (en) * | 1980-09-19 | 1989-11-07 | General Hospital Corporation | Immunoassay utilizing monoclonal high affinity IgM antibodies |
| DE3174064D1 (en) * | 1980-10-15 | 1986-04-17 | Takeda Chemical Industries Ltd | Method for immunochemical assay and kit therefor |
| US4837167A (en) * | 1981-01-30 | 1989-06-06 | Centocor, Inc. | Immunoassay for multi-determinant antigens using high-affinity |
| GB2118300B (en) * | 1982-02-12 | 1985-06-19 | Corning Glass Works | Method of immunoassay |
| DK76983A (en) * | 1982-03-05 | 1983-09-06 | Takeda Chemical Industries Ltd | METHOD AND REAGENT FOR IMMUNKEMIC DETERMINATION OF HUMAN CHORIOGONADOTROPIN |
| DE3225027A1 (en) * | 1982-07-05 | 1984-01-05 | Boehringer Mannheim Gmbh, 6800 Mannheim | IMMUNCHEMICAL MEASUREMENT METHOD |
| IL73938A (en) * | 1984-01-02 | 1989-09-28 | Boehringer Mannheim Gmbh | Process and reagent for the determination of polyvalent antigen by incubation with three different receptors |
| US4690890A (en) * | 1984-04-04 | 1987-09-01 | Cetus Corporation | Process for simultaneously detecting multiple antigens using dual sandwich immunometric assay |
| US5011771A (en) * | 1984-04-12 | 1991-04-30 | The General Hospital Corporation | Multiepitopic immunometric assay |
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| US5770459A (en) * | 1986-04-30 | 1998-06-23 | Igen International, Inc. | Methods and apparatus for improved luminescence assays using particle concentration, electrochemical generation of chemiluminescence detection |
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| US5935779A (en) * | 1988-11-03 | 1999-08-10 | Igen International Inc. | Methods for improved particle electrochemiluminescence assay |
| DE3851050T2 (en) * | 1987-08-25 | 1995-03-16 | Fuji Yakuhin Kogyo Kk | DIAGNOSTIC METHOD FOR CHRONIC JOINT RHEUMATISM. |
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| US5962218A (en) * | 1988-11-03 | 1999-10-05 | Igen International Inc. | Methods and apparatus for improved luminescence assays |
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| US5705402A (en) * | 1988-11-03 | 1998-01-06 | Igen International, Inc. | Method and apparatus for magnetic microparticulate based luminescence assay including plurality of magnets |
| US5779976A (en) * | 1988-11-03 | 1998-07-14 | Igen International, Inc. | Apparatus for improved luminescence assays |
| WO1990007114A1 (en) * | 1988-12-12 | 1990-06-28 | Commonwealth Serum Laboratories Commission | Solid phase immuno-assay with labelled conjugate |
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| ZA92804B (en) | 1991-02-06 | 1992-12-30 | Igen Inc | Methods and apparatus for improved luminescence assays |
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| FR2710410B1 (en) * | 1993-09-20 | 1995-10-20 | Bio Merieux | Method and device for determining an analyte in a sample. |
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| US6844163B1 (en) | 1999-04-12 | 2005-01-18 | Sumitomo Chemical Co., Ltd. | Method for analyzing the amount of intraabdominal adipose tissue |
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- 1981-04-14 DE DE19813115115 patent/DE3115115A1/en active Granted
- 1981-04-15 NL NLAANVRAGE8101860,A patent/NL187545B/en not_active Application Discontinuation
- 1981-04-22 SE SE8102558A patent/SE460930B/en not_active IP Right Cessation
- 1981-04-23 FR FR8108105A patent/FR2481318A1/en active Granted
- 1981-04-24 GB GB8112730A patent/GB2074727B/en not_active Expired
- 1981-04-24 AU AU69811/81A patent/AU542563B2/en not_active Expired
- 1981-04-24 NO NO811407A patent/NO159620C/en not_active IP Right Cessation
- 1981-04-24 AT AT0186481A patent/AT373398B/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| ATA186481A (en) | 1983-05-15 |
| DK151399C (en) | 1988-09-05 |
| NL8101860A (en) | 1981-11-16 |
| AU542563B2 (en) | 1985-02-28 |
| FR2481318A1 (en) | 1981-10-30 |
| NO811407L (en) | 1981-10-26 |
| DK155881A (en) | 1981-10-26 |
| DK151399B (en) | 1987-11-30 |
| GB2074727B (en) | 1983-11-30 |
| IT8121122A0 (en) | 1981-04-13 |
| AT373398B (en) | 1984-01-10 |
| NL187545B (en) | 1991-06-03 |
| GB2074727A (en) | 1981-11-04 |
| DE3115115C2 (en) | 1987-02-12 |
| SE460930B (en) | 1989-12-04 |
| IT1137344B (en) | 1986-09-10 |
| CA1160566A (en) | 1984-01-17 |
| DE3115115A1 (en) | 1982-02-04 |
| SE8102558L (en) | 1981-10-26 |
| NO159620C (en) | 1989-01-18 |
| AU6981181A (en) | 1981-10-29 |
| FR2481318B1 (en) | 1984-10-19 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MK1K | Patent expired |
Free format text: EXPIRED IN APRIL 2001 |