DK151399B - IMMUNOLOGICAL PROCEDURE FOR DETERMINING A SUBSTANCE BY COMPLETION WITH IMMUNOLOGICALLY ACTIVE PARTICIPANTS - Google Patents
IMMUNOLOGICAL PROCEDURE FOR DETERMINING A SUBSTANCE BY COMPLETION WITH IMMUNOLOGICALLY ACTIVE PARTICIPANTS Download PDFInfo
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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Description
i 151399in 151399
Den foreliggende opfindelse angår en enzymimmunfremgangsmåde ifølge det såkaldte "sandwich-princip". Ifølge dette princip omsættes det stof, der skal bestemmes, og som kan være et antigen, et antistof eller en hapten, med to immunologisk aktive reaktionsdeltagere. I 5 regelen er den ene af disse immunologisk aktive reaktionsdeltagere bundet til et vanduopløseligt bærestof, medens den anden er forsynet med et egnet enzym. I praksis omsættes det stof, der skal bestemmes, først med den på et bærestof bundne reaktionsdeltager, og efter faseadskillelse og vask omsættes det i mellemtiden på bærestoffet 10 immunologisk bundne stof med den anden, enzymmærkede reaktionsdeltager. Efter fornyet faseadskillelse foregår den enzymatiske reaktion til kvantitativ påvisning af det stof, der skal bestemmes, enten i fast eller i flydende fase.The present invention relates to an enzyme immune method according to the so-called "sandwich principle". According to this principle, the substance to be determined, which may be an antigen, an antibody or a hapten, is reacted with two immunologically active reaction participants. As a rule, one of these immunologically active reaction participants 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 reacted with the reaction participant bound to a carrier, and after phase separation and washing, the intermediate 10 immunologically bound substance is subsequently reacted with the other enzyme-labeled reaction participant. After renewed phase separation, the enzymatic reaction takes place to quantitatively detect the substance to be determined, either in solid or liquid phase.
I DE-OS 27 44 836 beskrives en immunokemisk målemetode, som er en 15 kombination af sandwich-metoden og den konkurrende metode, og som er ejendommelig ved, at efter omsætning mellem det antigen, der skal måles, og det insolubiliserede antistof til antigenet omsættes antige-net/antistof-complexet uden isolering fra reaktionsblandingen kontinuerligt med det markerede antistof.DE-OS 27 44 836 discloses an immunochemical measurement method which is a combination of the sandwich method and the competing method, which is characterized in that after conversion between the antigen to be measured and the insolubilized antibody to the antigen, it is reacted antigen-net / antibody complex without isolation from the reaction mixture continuously with the labeled antibody.
20 I DE-OS 29 25 565 beskrives en et-trins sandwich-metode, som eksemplificeres ved en radioimmuno-fremgangsmåde under anvendelse af polyklonale antistoffer. Anvendelsen af monoklonale antistoffer eller anvendelse af et monoklonalt antistof og et polyklonalt antistof samt anvendelsen af en enzymimmunofremgangsmåde gøres på ingen måde 25 nærliggende i dette offentliggørelsesskrift.DE-OS 29 25 565 discloses a one-step sandwich method exemplified by a radioimmunoassay using polyclonal antibodies. The use of monoclonal antibodies or the use of a monoclonal antibody and a polyclonal antibody, as well as the use of an enzyme immunoassay method, are by no means obvious in this disclosure.
Inden for rammerne af den foreliggende opfindelse har det overraskende nok nu vist sig, at man i modsætning til den tidligere opfattelse også kan arbejde med en "étkars-teknik", ved hvilken det stof, der skal bestemmes, omsættes samtidig med begge de immunologisk 30 aktive reaktionsdeltagere i en enkelt inkubering, når der som monologisk reaktionsdeltagere anvendes to forskellige monoklonale antistoffer eller et monoklonalt antistof og et antistof fra en anden dyreart.Surprisingly, within the scope of the present invention, it has now been found that, contrary to the foregoing, it is also possible to work with a "one-vessel technique" by which the substance to be determined is reacted simultaneously with both of the immunologically active reaction participants in a single incubation when, as monologic reaction participants, two different monoclonal antibodies or a monoclonal antibody and an antibody from another animal species are used.
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Den foreliggende opfindelse bygger på denne erkendelse og angår således en immunologisk fremgangsmåde til påvisning eller bestemmelse af et stof med en immunologisk aktiv reaktionsdeltager, som er forsynet med et enzym, samt med en immunologisk aktiv reaktionsdeltager, 5 som er bundet eller bindes til et vanduopløseligt bærestof, ved inku-bering af det stof, der skal bestemmes, med de immunologisk aktive reaktionsdeltagere, påfølgende adskillelse af fast og flydende fase og måling af enzymmærknrngens omfang, enten i den faste eller i den flydende fase, som et mål for mængden af det stof, der skal bestem-10 mes, hvilken fremgangsmåde er ejendommelig ved, at der som immunologisk aktive reaktionsdeltagere anvendes to forskellige monoklonale antistoffer eller et monoklonalt antistof og et antistof fra en anden dyreart, som inkuberes fra starten sammen med det stof, der skal bestemmes.The present invention is based on this disclosure and thus relates to an immunological method for detecting or determining a substance having an immunologically active reaction participant provided with an enzyme and to an immunologically active reaction participant which is bound or bound to a water-insoluble carrier. , by incubating the substance to be determined, with the immunologically active reaction participants, subsequent separation of solid and liquid phase, and measuring the extent of the enzyme labeling, either in the solid or in the liquid phase, as a measure of the amount of the substance to determine which method is peculiar to use as immunologically active reaction participants two different monoclonal antibodies or a monoclonal antibody and another antibody antibody which is initially incubated with the substance to be determined.
15 Som stof, der skal bestemmes, kan nævnes alle bestanddele i fysiologiske væsker, celle- eller vævsekstrakter, til hvilke der forekommer eller kan dannes immunologiske reaktionsdeltagere, og som har to eller flere immunologisk aktive positioner. Dertil hører peptider, proteiner, lipoproteiner, glycoproteiner, steroler, steroider, lipoider, nucleinsy-20 rer, enzymer, hormoner, polysaccharider og alkaloider. Foretrukne immunologisk aktive stoffer er de naturlige antigener såsom hormoner, enzymer, organspecifikke antigener, bindevævskomponenter, blodcelle-antigener, plasmaproteiner og patologiske globuliner. Særlig foretrukne stoffer er det carcinoembryoniske antigen (CEA) samt humant 25 choriongonadotropin (HCG).As a substance to be determined, all the constituents of physiological fluids, cell or tissue extracts to which there are or may be formed immunologic response participants and having two or more immunologically active positions may be mentioned. These include peptides, proteins, lipoproteins, glycoproteins, sterols, steroids, lipoids, nucleic acids, enzymes, hormones, polysaccharides and alkaloids. Preferred immunologically active substances are the natural antigens such as hormones, enzymes, organ specific antigens, connective tissue components, blood cell antigens, plasma proteins and pathological globulins. Particularly preferred substances are the carcinoembryonic antigen (CEA) and human chorionic gonadotropin (HCG).
Ved fremgangsmåden ifølge opfindelsen tjener den immunologisk aktive reaktionsdeltager, som er forsynet med et enzym, som indikator for den immunologiske reaktion. Foretrukne enzymer er den alkaliske phosphatase, malat-dehydrogenasen, glucose-6-phosphat-dehydrogena-30 sen, glucose-oxidasen, glucoamylasen, galactosidasen og acetylcholin-esterasen. En særlig foretrukken enzym-label er peroxidase fra peberrod.In the method of the invention, the immunologically active reaction participant, provided with an enzyme, serves as an indicator of the immunological response. Preferred enzymes are the alkaline phosphatase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase, galactosidase and acetylcholine esterase. A particularly preferred enzyme label is horseradish peroxidase.
Enzymet som indikator for den immunologiske reaktion måles i den flydende, men fortrinsvis i den faste fase ved kendte metoder og er 3 151399 et mål for mængden af det stof, der skal bestemmes. Ved peroxidase fra perberrod som label måles enzymmængden fortrinsvis på grundlag af den iboende katalytiske aktivitet, som bestemmes ved hjælp af og o-phenylendiamin som redoxindikator. Efter 30 minutters katalytisk 5 reaktion måles farveintensiteten i den oxiderede redoxindikator fotometrisk.The enzyme as indicator of the immunological response is measured in the liquid but preferably in the solid phase by known methods and is a measure of the amount of the substance to be determined. For peroxidase from Perberrod as a label, the amount of enzyme is preferably measured on the basis of the inherent catalytic activity determined by and o-phenylenediamine as a redox indicator. After 30 minutes of catalytic reaction, the color intensity of the oxidized redox indicator is measured photometrically.
Den anden immunologisk aktive reaktionsdeltager tjener til fraskillelse af det stof, der skal bestemmes, fra analyseprøven. Til dette adskillelsestrin kan den anden immunologisk aktive reaktionsdeltager fra 10 starten være bundet til et vanduopløseligt bærestof, eller det kan bindes til et relevant bærestof under eller efter den immunologiske reaktion.The other immunologically active reaction participant serves to separate the substance to be determined from the assay. For this separation step, the second immunologically active reaction participant may, from the start, be bound to a water-insoluble carrier, or it may be bound to a relevant carrier during or after the immunological reaction.
Som vanduopløselige bærestoffer for den anden immunologisk aktive reaktionsdeltager kan f.eks. nævnes: organiske og uorganiske polyme-15 rer (amylase, dextraner, nativ eller modificeret cellulose, polyacryl-amid, agarose, magnetit, porøst glaspulver, polyvinylidenfluorid og latex), indervæggen i testbeholdere (reagensglas, titrerplader eller kuvetter af glas eller kunststof) samt overfladen af faste stoffer (glas- og kunststofstave, stave med fortykkelse for enden og stave 20 med vinger eller lameller for enden). Særlig egnede bærestoffer til fremgangsmåden ifølge opfindelsen er glas- og kunststofkugler.As water-insoluble carriers for the other immunologically active reaction participant, e.g. Mention is made of: organic and inorganic polymers (amylase, dextrans, native or modified cellulose, polyacrylamide, agarose, magnetite, porous glass powder, polyvinylidene fluoride and latex), inner wall of test containers (test tubes, titer plates or cuvettes) and the surface of solids (glass and plastic rods, thickened rod at the end and rod 20 with wings or slats at the end). Particularly suitable carriers for the process according to the invention are glass and plastic balls.
Den anden immunologisk aktive reaktionsdeltager kan være fysisk (adsorptivt) eller kemisk bundet til det vanduopløselige bærestof, eller det kan ved hjælp af en yderligere reaktionsdeltager, der på sin 25 side er bundet til et bærestof, bindes under eller efter reaktionen.The other immunologically active reaction participant may be physically (adsorptively) or chemically bound to the water-insoluble carrier, or it may be bound during or after the reaction by an additional reaction participant, which is in turn bound to a carrier.
Det er væsentligt for fremgangsmåden ifølge opfindelsen, at det stof, der skal bestemmes, har mindst to immunologisk aktive positioner (epitoper), som kan erkendes af de to immunologisk aktive reaktionsdeltagere, den til et bærestof bundne og den med et enzym forsynede 30 reaktionsdeltager, og kan omsættes. Som immunologisk aktive reaktionsdeltagere benyttes to forskellige monoklonale antistoffer eller et monoklonalt antistof og et antistof fra en anden dyreart, som ganske vist begge reagerer med det stof, der skal bestemmes, men som hver 4 151399 er rettet mod forskellige immunologisk aktive positioner. Til bestemmelse af antigener er det særlig velegnet med en kombination af antistoffer fra forskellige kloner eller af monoklonale antistoffer og antistoffer fra en anden dyreart.It is essential for the method of the invention that the substance to be determined has at least two immunologically active positions (epitopes) which can be recognized by the two immunologically active reaction participants, the carrier bound and the enzyme-associated reaction participant. and can be traded. As immunologically active reaction participants, two different monoclonal antibodies or a monoclonal antibody and an antibody from another animal species are used, both of which react with the substance to be determined, but each targeting different immunologically active positions. For the determination of antigens, it is particularly suitable with a combination of antibodies from different clones or of monoclonal antibodies and antibodies from another animal species.
5 Til den immunologiske reaktion kan prøven med det stof, der skal bestemmes, anvendes direkte eller fortyndet eller forbehandlet på egnet måde. Til forbehandling kan det stof, der skal bestemmes, isoleres, beriges eller befries for forstyrrende bestanddele.5 For the immunological reaction, the sample with the substance to be determined can be used directly or diluted or pretreated in a suitable manner. For pretreatment, the substance to be determined can be isolated, enriched or freed from disruptive constituents.
Ved fremgangsmåden ifølge opfindelsen omsættes det stof, der skal 10 bestemmes, samtidig med den enzymmærkede og den bærerbundne immunologisk aktive reaktionsdeltager. Rækkefølgen af tilsætningen af reagenserne retter sig efter arten af det valgte bærestof sy stem. Ved arbejde med en sensibiliseret plastkugle sammenhældes først den enzymmærkede reaktionsdeltager og det stof, der skal bestemmes, i et 15 egnet reagensglas, hvorefter den med den anden reaktionsdeltager sensibiliserede kugle tilsættes.In the method of the invention, the substance to be determined is reacted simultaneously with the enzyme-labeled and the carrier-bound immunologically active reaction participant. The order of addition of the reagents depends on the nature of the selected carrier system. When working with a sensitized plastic ball, the enzyme labeled reaction participant and the substance to be determined are first bonded into a suitable test tube, after which the sensitized ball with the other reaction participant is added.
Den immunologiske reaktion udføres i et egnet puffersystem for at opretholde en optimal pH-værdi, der kan ligge mellem 4 og 9. Foretrukne puffere er f.eks. acetatpuffer, citratpuffer, phosphatpuffer, 20 tris-puffer, triethanolaminpuffer, boratpuffer og glycinpuffer. Der kan også anvendes pufferblandinger.The immunological reaction is carried out in a suitable buffer system to maintain an optimum pH value which may be between 4 and 9. Preferred buffers are e.g. acetate buffer, citrate buffer, phosphate buffer, tris buffer, triethanolamine buffer, borate buffer and glycine buffer. Buffer mixtures may also be used.
Den immunologiske reaktion foretages fortrinsvis ved en temperatur på mellem 0 og 55°C. Normalt tiltager den immunologiske reaktionshastighed med højere temperaturer, hvorved ligevægten hurtigere nås under 25 ellers samme testbetingelser.The immunological reaction is preferably carried out at a temperature of between 0 and 55 ° C. Normally, the immunological reaction rate increases with higher temperatures, thus reaching equilibrium more quickly under otherwise the same test conditions.
Inkuberingen af det stof, der skal bestemmes, med den enzymmærkede reaktionsdeltager og den bærerbundne reaktionsdeltager kan foretages, indtil ligevægten er nået. Den immunologiske reaktion kan dog også afbrydes på et tidligere tidspunkt, idet den faste og den fly-30 dende fase adskilles efter en bestemt inkubationstid, og enzymmærkningens omfang bestemmes enten i den flydende eller i den faste fase.The incubation of the substance to be determined with the enzyme-labeled reaction participant and the carrier-bound reaction participant can be carried out until the equilibrium is reached. However, the immunological reaction may also be interrupted at an earlier stage, separating the solid and liquid phase after a certain incubation time, and the extent of enzyme labeling is determined either in the liquid or in the solid phase.
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Ved den immunologiske reaktion kan der træffes foranstaltninger til stabilisering af den immunologiske aktivitet af reaktionsdeltagerne og af det stof, der skal bestemmes, samt af enzymet. Endvidere kan der til inkubationsopløsningen sættes bestanddele, f.eks. proteiner og 5 detergenter, for at udelukke uspecifikke reaktioner, til formindskelse af hæmmende indflydelser eller til aktivering.In the immunological reaction, measures can be taken to stabilize the immunological activity of the reaction participants and of the substance to be determined as well as of the enzyme. Furthermore, ingredients can be added to the incubation solution, e.g. proteins and 5 detergents, to exclude nonspecific reactions, to reduce inhibitory influences or to activate.
Fremgangsmåden ifølge den foreliggende opfindelse er overordentlig følsom og udmærker sig ved at være enkel at håndtere.The process of the present invention is extremely sensitive and distinguishes itself by being simple to handle.
Fremgangsmåden ifølge opfindelsen belyses nærmere ved nedenstående 10 eksempler:The process according to the invention is further illustrated by the following examples:
Eksempel 1.Example 1.
Kvantitativ bestemmelse af CEA i patientplasma med et monoklonalt CEA-antistof og et anvendeligt CEA-antistof (ged).Quantitative determination of CEA in patient plasma with a monoclonal CEA antibody and a useful CEA antibody (goat).
I hvert af det nødvendige antal reagensglas (10 x 75 mm) afpipetteres 15 0,2 ml testopløsning (0,2 mol/liter Ν3Η2ΡΟ^/Ν82ΗΡΟ^, pH-værdi 6,5, med 2 g/liter kvægserumalbumin, 20%'s normalt gedeserum og 0,2 vg/ml gede-anti-CEA-peroxidase-konjugat), 0,050 ml af det patientplasma, der skal analyseres, henholdsvis CEA-standarder (0 ng/ml CEA, 2,5 ng/ml CEA, 10 ng/ml CEA og 20 ng/ml CEA) og CEA-kon-20 trolserum (5,0 ng/ml CEA ± 1,0 ng/ml) tilsættes, og til hvert glas sættes en med monoklonalt muse-anti-CEA sensibiliseret polystyrenkugle* (ø = 6,5 mm), og der inkuberes i 16 timer ved 37°C. Derefter vaskes polystyren kuglerne tre gange med hver gang 2 - 5 ml destilleret vand, hver overføres til 0,5 ml substratpuffer til aktivitetsbestem-25 melse af peroxidasen (0,1 mol/liter kaliumcitratpuffer med pH-værdi 5,0 med 6 mmol/liter og 20 mmol/liter o-phenylendiamin) og inkuberes i 30 minutter ved stuetemperatur (22°C). Til afbrydelse af den peroxidatiske aktivitet samt til intensivering af fa rvei nten siteten tilsættes 2,0 ml IN HCI, og inden for 30 minutter måles ekstinktionen 30 fotometrisk ved en bølgelængde på 492 nm. I tabel I er værdierne for en CEA-bestemmelse anført og sammenlignet med de værdier, der er opnået med Roche's radioimmunoassay.In each of the required number of test tubes (10 x 75 mm), pipette 0.2 ml of test solution (0.2 mol / liter Ν3 g2ΡΟ ^ / Ν82ΗΡΟ,, pH 6.5, with 2 g / liter bovine serum albumin, 20%). s normal goat serum and 0.2 vg / ml goat anti-CEA peroxidase conjugate), 0.050 ml of the patient plasma to be analyzed, respectively CEA standards (0 ng / ml CEA, 2.5 ng / ml CEA, 10 ng / ml CEA and 20 ng / ml CEA) and CEA control serum (5.0 ng / ml CEA ± 1.0 ng / ml) are added and to each glass one with anti-CEA monoclonal mouse is added. sensitized polystyrene ball * (ø = 6.5 mm) and incubated for 16 hours at 37 ° C. Then, the polystyrene beads are washed three times with 2 - 5 ml of distilled water each time, each transferred to 0.5 ml of substrate buffer for activity determination of the peroxidase (0.1 mol / liter potassium citrate buffer of pH 5.0 with 6 mmol / and 20 mmol / liter o-phenylenediamine) and incubated for 30 minutes at room temperature (22 ° C). To discontinue the peroxidative activity and to intensify the site, add 2.0 ml of 1N HCl and within 30 minutes the extinction 30 is measured photometrically at a wavelength of 492 nm. In Table I, the values of a CEA determination are listed and compared with those obtained with Roche's radioimmunoassay.
6 151399 * Fremstillingen af monoklonalt muse-anti-CEA foretages i analogi med den i Journal of Immunological Methods, 32 (1980) 297-304 beskrevne metode, idet der som udgangscellelinje til fusionen anvendes myelom-linjen Sp 2/01-AG, der er deponeret i ATCC under nr. CRL 8006.The preparation of mouse anti-CEA monoclonal is done by analogy to the method described in Journal of Immunological Methods, 32 (1980) 297-304, using as the starting cell line for the fusion the myeloma line Sp 2/01-AG which is deposited in the ATCC under No. CRL 8006.
5 Fusionen foretages med miltceller fra mus, som er blevet immuniseret med CEA. Immuniseringen af musene blev foretaget i analogi med tabel 1 i den nævnte publikation, idet de første to immuniseringer hver blev foretaget med 50 ug CEA, immunisering 3 og 4 blev udeladt, immunisering 5 blev foretaget med 50 ug CEA og immunisering 6-8 10 hver med 200 ug CEA.5 The fusion is performed with spleen cells from mice that have been immunized with CEA. Immunization of the mice was done by analogy with Table 1 of the said publication, the first two immunizations each being made with 50 µg of CEA, immunizations 3 and 4 being omitted, immunization 5 being done with 50 µg of CEA, and immunization 6-8 each with 200 µg CEA.
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Tabel ITable I
Prøvemateriale iE492 nn,/RT/30 Bin- CEA-Standard O ng/ml CEA 0,103 2,5 ng/ml CEA 0,330 10.0 ng/ml CEA 0,978 20.0 ng/ml CEA 1,850 CEA- kontrolserum 5,0 ng/ml CEA 0,540Test material iE492 nn, / RT / 30 Bin CEA Standard O ng / ml CEA 0.103 2.5 ng / ml CEA 0.330 10.0 ng / ml CEA 0.978 20.0 ng / ml CEA 1.850 CEA Control Serum 5.0 ng / ml CEA 0.540
Patientplasma ROCHE-RIA-Test Fremgangsmåden ifølge opfindelsen_Patient Plasma ROCHE-RIA Test The method of the invention
Nr. 7212 0,6 ng/ml 1,2 ng/mlNo. 7212 0.6 ng / ml 1.2 ng / ml
Nr. 7188 2,2 ng/ml 1,0 ng/mlNo. 7188 2.2 ng / ml 1.0 ng / ml
Nr. 7220 1,2 ng/ml 1,4 ng/ml I Nr. 7218 1,2 ng/ml 1,3 ng/ml ; Nr. 7234 2,5 ng/ml 2,7 ng/ml i Nr. 7249 2,4 ng/ml 2,0 ng/mlNo. 7220 1.2 ng / ml 1.4 ng / ml I Nr. 7218 1.2 ng / ml 1.3 ng / ml; No. 7234 2.5 ng / ml 2.7 ng / ml in Nr. 7249 2.4 ng / ml 2.0 ng / ml
Nr. 7203 2,3 ng/ml 2,0 ng/mlNo. 7203 2.3 ng / ml 2.0 ng / ml
Nr. 7223 3,0 ng/ml 3,3 ng/mlNo. 7223 3.0 ng / ml 3.3 ng / ml
Nr. 7247 3,1 ng/ml 2,8 ng/mlNo. 7247 3.1 ng / ml 2.8 ng / ml
Nr. 7258 4,6 ng/ml 4,3 ng/mlNo. 7258 4.6 ng / ml 4.3 ng / ml
Nr. 7215 4,9 ng/ml 5,5 ng/mlNo. 7215 4.9 ng / ml 5.5 ng / ml
Nr. 7219 5,0 ng/ml 5,9 ng/mlNo. 7219 5.0 ng / ml 5.9 ng / ml
Nr. 8180 8,6 ng/ml 8,5 ng/mlNo. 8180 8.6 ng / ml 8.5 ng / ml
Nr. 7248 11,2 ng/ml 10,7 ng/mlNo. 7248 11.2 ng / ml 10.7 ng / ml
Nr. 7262 14,2 ng/ml 14,3 ng/mlNo. 7262 14.2 ng / ml 14.3 ng / ml
Nr. 7201 15,6 ng/ml 15,3 ng/ml Værdier på under 2,5 ng/ml CEA ligger i normalområdet, medens værdier på over 2,5 ng/ml ligger i det patologiske område.No. 7201 15.6 ng / ml 15.3 ng / ml Values below 2.5 ng / ml CEA are in the normal range, while values above 2.5 ng / ml are in the pathological range.
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Eksempel 2.Example 2.
Kvantitativ bestemmelse af CEA i patientplasma med monoklonale CEA-antistoffer fra to forskellige kloner.Quantitative determination of CEA in patient plasma with monoclonal CEA antibodies from two different clones.
I hvert af det nødvendige antal reagensglas (10 x 75 mm) afpipetteres 5 0,2 ml testopløsning (0,2 mol/liter Naf^PO^/ts^HPO^, pH-værdi 6,5, med 2 g/liter kvaegserumalbumin, 20%'s normalt gedeserum og 0,15 μg/ml monoklonalt muse-anti-CEA-peroxidase-konjugat0), 0,050 ml af det patientplasma, der skal analyseres, henholdsvis CEA-standarder (0 ng/ml CEA, 2,5 ng/ml CEA, 10 ng/ml CEA og 20 ng/ml CEA) og 10 CEA-kontrolserum (5,0 ng/ml CEA ±1,0 ng/ml) tilsættes, og til hvert glas sættes en med monoklonalt muse-anti-CEA sensibiliseret polystyrenkugle1 (ø = 6,5 mm), og der inkuberes i 16 timer ved 37°C. Derefter vaskes polystyren kuglerne tre gange med hver gang 2 - 5 ml destilleret vand, hver overføres til 0,5 ml substratpuffer til aktivi-15 tetsbestemmelse af peroxidasen (0,1 mol/liter kaliumcitratpuffer med pH-værdi 5,0 med 6 mmol/liter og 20 mmol/liter o-phenylendiamin) og inkuberes i 30 minutter ved stuetemperatur (22°C). Til afbrydelse af den peroxidatiske aktivitet samt til intensivering af farveintensite-ten tilsættes 2,0 ml 1N HCI, og inden for 30 minutter måles ekstinktio-20 nen fotometrisk ved en bølgelængde på 492 nm. I tabel III er værdierne for en CEA-bestemmelse anført og sammenlignet med de værdier, der er opnået med Roche's radioimmunoassay.In each of the required number of tubes (10 x 75 mm), pipette 5 0.2 ml of test solution (0.2 mol / liter Naf ^ PO ^ / ts ^ HPO ^, pH 6.5, with 2 g / liter bovine serum albumin , 20% normal goat serum and 0.15 μg / ml mouse anti-CEA peroxidase conjugate0), 0.050 ml of the patient plasma to be analyzed, respectively CEA standards (0 ng / ml CEA, 2.5 ng / ml CEA, 10 ng / ml CEA and 20 ng / ml CEA) and 10 CEA control serum (5.0 ng / ml CEA ± 1.0 ng / ml) are added, and to each glass one with mouse monoclonal anti-CEA sensitized polystyrene ball1 (ø = 6.5 mm) and incubated for 16 hours at 37 ° C. Then, the polystyrene beads are washed three times with 2 - 5 ml of distilled water each time, each transferred to 0.5 ml of substrate buffer for activity determination of the peroxidase (0.1 mole / liter of potassium citrate buffer of 5.0 with 6 mmol / and 20 mmol / liter o-phenylenediamine) and incubated for 30 minutes at room temperature (22 ° C). To interrupt the peroxidative activity and to intensify the color intensity, 2.0 ml of 1N HCl is added and within 30 minutes the extinct ion is measured photometrically at a wavelength of 492 nm. Table III lists the values of a CEA assay and compares it with the values obtained with Roche's radioimmunoassay.
Fremstillingen af monoklonalt muse-anti-CEA foretages i analogi med den i Journal of Immunological Methods, 32 (1980) 297-304 beskrevne 25 metode, idet der som udgangscellelinje til fusionen anvendes myelom-linjen Sp 2/01-AG, der er deponeret i ATCC under nr. CRL 8006.The preparation of mouse anti-CEA monoclonal is done by analogy to the method described in Journal of Immunological Methods, 32 (1980) 297-304, using as the starting cell line for the fusion, the myeloma line Sp 2/01-AG deposited in ATCC under No. CRL 8006.
Fusionen foretages med miltceller fra mus, som er blevet immuniseret med CEA. Immuniseringen af musene blev foretaget i analogi med tabel 1 i den nævnte publikation, idet de første to immuniseringer hver 30 blev foretaget med 50 μg CEA, immunisering 3 og 4 blev udeladt, immunisering 5 blev foretaget med 50 μg CEA og immunisering 6-8 hver med 200 μg CEA. Der anvendes to forskellige, egnede monoklonale antistoffer, som er rettet mod forskellige epitoper i CEA-antige-net.The fusion is performed with spleen cells from mice that have been immunized with CEA. Immunization of the mice was done by analogy with Table 1 of the aforementioned publication, the first two immunizations each being made with 50 µg CEA, immunizations 3 and 4 omitted, immunization 5 being done with 50 µg CEA, and immunization 6-8 each with 200 µg CEA. Two different suitable monoclonal antibodies are used which target different epitopes in CEA antigen networks.
9 1513999 151399
Tabel Ij ΔΕTable Ij ΔΕ
Prøvemateriale 492 nm/RT/30 min.Sample material 492 nm / RT / 30 min.
CEA-Standard O ng/ml CEA 0,390 2,5 ng/ml CEA 0,440 10.0 ng/ml CEA 0,620 20.0 ng/ml CEA 0,860 . 1 1 CEA-kontrolserum 5,0 ng/ml CEA 0,510CEA Standard O ng / ml CEA 0.390 2.5 ng / ml CEA 0.440 10.0 ng / ml CEA 0.620 20.0 ng / ml CEA 0.860. 1 1 CEA Control Serum 5.0 ng / ml CEA 0.510
Patientplasma ROCHE-RIA-Tes t Fremgangsmåden ifølge opfindelsen._Patient Plasma ROCHE-RIA-Tes t The method of the invention.
Nr. 7220 1,2 ng/ml 0,8 ng/mlNo. 7220 1.2 ng / ml 0.8 ng / ml
Nr. 7234 2,5 ng/ml 3,0 ng/mlNo. 7234 2.5 ng / ml 3.0 ng / ml
Nr. 7223 3,0 ng/ml 3,5 ng/mlNo. 7223 3.0 ng / ml 3.5 ng / ml
Nr. 7247 3,1 ng/ml 3,2 ng/mlNo. 7247 3.1 ng / ml 3.2 ng / ml
Nr. 7258 4,6 ng/ml 4,4 ng/mlNo. 7258 4.6 ng / ml 4.4 ng / ml
Nr. 7215 4,9 ng/ml 5,0 ng/mlNo. 7215 4.9 ng / ml 5.0 ng / ml
Nr. 7219 5,0 ng/ml 5,4 ng/mlNo. 7219 5.0 ng / ml 5.4 ng / ml
Nr. 8180 8,6 ng/ml 8,6 ng/mlNo. 8180 8.6 ng / ml 8.6 ng / ml
Nr. 7248 11,2 ng/ml 11,0 ng/mlNo. 7248 11.2 ng / ml 11.0 ng / ml
Nr> 7262 14,2 ng/ml 14.0 ng/mlNo.> 7262 14.2 ng / ml 14.0 ng / ml
Nr. 7201 15,6 ng/ml 16,0 ng/ml j i _____: Værdier på under 2,5 ng/ml CEA ligger i normalområdet, medens værdier på over 2,5 ng/ml ligger i det patologiske område.No. 7201 15.6 ng / ml 16.0 ng / ml j in _____: Values below 2.5 ng / ml CEA are in the normal range, while values greater than 2.5 ng / ml are in the pathological range.
151399 ίο151399 ίο
Eksempel 3.Example 3
Kvantitativ bestemmelse af HCG i serum/plasma/urin.Quantitative determination of HCG in serum / plasma / urine.
I hvert af det nødvendige antal reagensglas (10 x 75 mm) afpipetteres 0,2 ml testopløsning (0,1 mol/liter Nah^PO^/I^HRO^, pH-værdi 7,0, 5 med 2 g/liter kvaegserumalbumin og 1,0 yg/ml monoklonalt muse-anti-HCG-peroxidase-konjugat°), 0,050 ml af det patientplasma, der skal analyseres, henholdsvis HCG-standarder (0, 25, 50, 100 og 250 mlU/ml HCG) tilsættes, og til hvert glas sættes en med kanin-anti-HCG sensibiliseret polystyren kugle (ø = 6,5 mm), og der inkuberes i 16 10 timer ved stuetemperatur i vandmættet atmosfære. Derefter vaskes polystyrenkuglerne tre gange med hver gang 2 - 5 ml destilleret vand, hver overføres til 0,5 ml substratpuffer til aktivitetsbestemmelse af peroxidasen (0,1 mol/liter kaliumcitratpuffer med pH-værdi 5,0 med 6 mmol/liter og 20 mmol/liter o-phenylendiamin) og inkube- 15 res i 30 minutter ved stuetemperatur (16 - 30°C). Til afbrydelse af den peroxidatiske aktivitet samt til intensivering af fa rvei nten siteten tilsættes 2,0 ml IN HCI, og inden for 30 minutter måles ekstinktionen fotometrisk ved en bølgelængde på 492 nm. I tabellerne er værdierne fra en HCG-bestemmelse i serum og i urin anført. 1 * Fremstillingen af monoklonalt anti-HCG kan foretages efter en af de i Journal of Immunogical Methods 32 (1980) 297 - 304 beskrevne metoder.In each of the required number of test tubes (10 x 75 mm), pipette 0.2 ml of test solution (0.1 mol / liter Nah ^ PO ^ / l ^ HRO ^, pH 7.0, 5 with 2 g / liter bovine serum albumin and 1.0 µg / ml mouse anti-HCG peroxidase conjugate (monoclonal mouse), 0.050 ml of the patient plasma to be analyzed, HCG standards (0, 25, 50, 100 and 250 mlU / ml HCG, respectively) are added. , and to each glass is added a rabbit anti-HCG sensitized polystyrene ball (λ = 6.5 mm) and incubated for 16 hours at room temperature in a water saturated atmosphere. Then, the polystyrene balls are washed three times with 2 - 5 ml of distilled water each time, each transferred to 0.5 ml of substrate buffer for activity determination of the peroxidase (0.1 mol / liter potassium citrate buffer of pH 5.0 with 6 mmol / liter and 20 mmol / liter of o-phenylenediamine) and incubated for 30 minutes at room temperature (16-30 ° C). To stop the peroxidative activity and to intensify the feathering site, add 2.0 ml of 1N HCl and within 30 minutes the extinction is measured photometrically at a wavelength of 492 nm. The tables show the values of a HCG determination in serum and in urine. 1 * The preparation of anti-HCG monoclonal can be done by one of the methods described in Journal of Immunogical Methods 32 (1980) 297-304.
11 151399 HCG-Bestemmelse i serum og i urin.11 151399 HCG Determination in Serum and in Urine.
1. Standardkurver.1. Standard curves.
1 iE492 nm/3° min-/RT1 iE492 nm / 3 ° min / RT
mlU HCG/ml Serum__Urin 0 0,185 0,385 25 0,385 0,525 50 0,530 0,720 100 0,830 1,05 }_250 0,185 1,59 2. Patientprøver.mlU HCG / ml Serum__Urin 0 0.185 0.385 25 0.385 0.525 50 0.530 0.720 100 0.830 1.05} _250 0.185 1.59 2. Patient samples.
Prøve nr. ^E492 nn/3l~ min -/RT = HCG/ml prøveSample No. ^ E492 nn / 3l ~ min - / RT = HCG / ml sample
Urin N-1032 E 0,375 0 " 58-418 0,575 (x 50) * 1500 " 58414 0,775 (x 100) * 5500 " 58416 0,810 (x 500) * 320.00Urine N-1032 E 0.375 0 "58-418 0.575 (x 50) * 1500" 58414 0.775 (x 100) * 5500 "58416 0.810 (x 500) * 320.00
Serum 2559 0,155 0 " 2560 0,125 0 " 1543 0,195 1,5 " 2673 0,505 44 " 1167 1,085 181 " 1492 0,98 (x 10) * 1370 " 2793 0,77 (x 20) * 1740 " 924 0,69 (x 100) * 7300 * Fortynding af prøvenSerum 2559 0.155 0 "2560 0.125 0" 1543 0.195 1.5 "2673 0.505 44" 1167 1.085 181 "1492 0.98 (x 10) * 1370" 2793 0.77 (x 20) * 1740 "924 0.69 ( x 100) * 7300 * Dilution of the sample
Omregning: 1 ng rent HCG svarer til ca. 10 mlU HCG.Conversion: 1 ng of pure HCG equals approx. 10 ml of HCG.
12 15139912 151399
Eksempel 4.Example 4
Kvantitativ bestemmelse af HCG i serum med monoklonale HCG-anti-stoffer fra to forskellige kloner.Quantitative determination of serum HCG with monoclonal HCG antibodies from two different clones.
I hvert af det nødvendige antal reagensglas (10 x 75 mm) afpipetteres 5 0,2 ml testopløsning (0,1 mol/liter Na^PO^/I^HPO^, pH-værdi 7,0, med 2 g/liter kvægserumalbumin og 1,0 ng/ml monoklonalt muse-an-ti-HCG-peroxidase-konjugat1), 0,050 ml af det patientplasma, der skal analyseres, henholdsvis HCG-standarder (0, 25, 50, 100 og 200 mlU/ml HCG) tilsættes, og til hvert glas sættes en med monoklonalt 10 muse-anti-HCG sensibiliseret polystyren kugle1 (ø = 6,5 mm), og der inkuberes f.eks. i 2 timer ved 37°C. Derefter vaskes polystyrenkuglerne tre gange med hver gang 2 - 5 ml destilleret vand, hver overføres til 0,5 ml substratpuffer til aktivitetsbestemmelse af peroxidasen (0,1 mol/liter kaliumcitratpuffer med pH-værdi 5,0 med 6 mmol/liter 15 P12^2 ^ mmol/liter o-phenylendiamin) og inkuberes i 30 minutter ved stuetemperatur (16 - 30°C). Til afbrydelse af den peroxidatiske aktivitet samt til intensivering af farveintensiteten tilsættes 1,0 ml 2N HCI, og inden for 30 minutter måles ekstinktionen fotometrisk ved en bølgelængde på 492 nm. I tabellerne er værdierne fra HCG-bestemmel-20 ser i serum anført.In each of the required number of test tubes (10 x 75 mm), pipette 5 0.2 ml of test solution (0.1 mol / liter Na 2 PO 2/1 H HPO 2, pH 7.0, with 2 g / liter bovine serum albumin and 1.0 ng / ml mouse monoclonal anti-HCG peroxidase conjugate1), 0.050 ml of the patient plasma to be analyzed, and HCG standards respectively (0, 25, 50, 100 and 200 mlU / ml HCG) is added and to each glass is added a monoclonal 10 mouse anti-HCG sensitized polystyrene ball1 (λ = 6.5 mm) and incubated, e.g. for 2 hours at 37 ° C. Then, the polystyrene balls are washed three times with 2 - 5 ml of distilled water each time, each transferred to 0.5 ml of substrate buffer for activity determination of the peroxidase (0.1 mole / liter of potassium citrate buffer of pH 5.0 with 6 mmol / liter of 15 P12 2 µmol / liter o-phenylenediamine) and incubated for 30 minutes at room temperature (16 - 30 ° C). To stop the peroxidative activity and to intensify the color intensity, 1.0 ml of 2N HCl is added and within 30 minutes the extinction is measured photometrically at a wavelength of 492 nm. The tables show the values from serum HCG determinations.
Fremstillingen af monoklonalt muse-HCG-antistof foretages efter den i Journal of Immunological Methods, 32 (1980) 297-304 beskrevne metode. Der anvendes to forskellige, egnede monoklonale antistoffer, som er rettet mod forskellige epitoper i HCG-antigenet.The preparation of mouse HCG antibody monoclonal antibody is performed according to the method described in Journal of Immunological Methods, 32 (1980) 297-304. Two different suitable monoclonal antibodies are used which target different epitopes of the HCG antigen.
13 151399 HCG-Bestemmelse i humant serum (middelværdier af dobbeltbestemmelser).13 151399 HCG Determination in Human Serum (Dual-Determination Values).
1. Standardkurver.1. Standard curves.
i [i ii —i [i ii -
ΔΕ 432 nm/30 min./RTΔΕ 432 nm / 30 min./RT
mu/ml HCG 1) Serum 2) Plasma 3) Urin 4) Puffer 0 0,038 0,054 0,158 0,180 25 0,226 - - - 50 0,475 0,544 0,557 0,716 100 0,905 0,955 0,970 1,40 200 1,75 1,55 1,90 2,44 i___- HCG-Værdierne i de nedenfor anførte eksempler på patientsera er 5 aflæst fra standardkurve 1). Standardkurverne 2), 3) og 4) er opstillet en anden dag med nye HCG-standarder.mu / ml HCG 1) Serum 2) Plasma 3) Urine 4) Buffer 0 0.038 0.054 0.158 0.180 25 0.226 - - 50 0.475 0.544 0.557 0.716 100 0.905 0.955 0.970 1.40 200 1.75 1.55 1.90 2, 44 of the ___ HCG values in the examples of patient sera listed below are 5 read from standard curve 1). The standard curves 2), 3) and 4) are set another day with new HCG standards.
2. Patientsera.2. Patient sera.
ΔΕ492/30 min./RT mIU/ml HCG Målt fra kurve l)ΔΕ492 / 30 min / RT mIU / ml HCG Measured from curve l)
Pool 091080 0,041 0Pool 091080 0.041 0
Nr. 2801 0,025 0No. 2801 0.025 0.
Nr. 3881 0,450 47No. 3881 0.450 47
Nr. 2673 1,13 127No. 2673 1.13 127
Nr. 1167 2,12 (1:2) 470No. 1167 2.12 (1: 2) 470
Nr. 4891 0,975 (1:50) 5*450No. 4891 0.975 (1:50) 5 * 450
Nr. 3240 1,06 (1:20) 2*280No. 3240 1.06 (1:20) 2 * 280
Nr. 4418 1,475 ( 1:1000) 167Ό00No. 4418 1,475 (1: 1000) 167Ό00
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| CH320980A CH642458A5 (en) | 1980-04-25 | 1980-04-25 | Immunological method |
| CH589880 | 1980-08-04 | ||
| CH589880A CH651396A5 (en) | 1980-08-04 | 1980-08-04 | Immunological method for detecting and determining carcinoembryonic antigen |
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| DK155881A DK155881A (en) | 1981-10-26 |
| DK151399B true DK151399B (en) | 1987-11-30 |
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| EP0146654A3 (en) * | 1980-06-20 | 1986-08-20 | Unilever Plc | Processes and apparatus for carrying out specific binding assays |
| US6406920B1 (en) | 1980-06-20 | 2002-06-18 | Inverness Medical Switzerland Gmbh | Processes and apparatus for carrying out specific binding assays |
| 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 |
| US4935339A (en) * | 1985-05-07 | 1990-06-19 | Nichols Institute Diagnostics | Delayed solid phase immunologic assay |
| 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 |
| DE3638767A1 (en) * | 1986-11-13 | 1988-05-26 | Behringwerke Ag | INCUBATION MEDIUM CONTAINING LACTOTRINE FOR SOLID-PHASE IMMUNOMETRIC METHOD AND ITS USE |
| US5935779A (en) * | 1988-11-03 | 1999-08-10 | Igen International Inc. | Methods for improved particle electrochemiluminescence assay |
| US6881589B1 (en) | 1987-04-30 | 2005-04-19 | Bioveris Corporation | Electrochemiluminescent localizable complexes for assay compositions |
| WO1989002078A1 (en) * | 1987-08-25 | 1989-03-09 | Fuji Yakuhin Kogyo Kabushiki Kaisha | Method for diagnosing chronic articular rheumatism |
| US5175084A (en) * | 1987-10-30 | 1992-12-29 | Fuji Yakuhin Kogyo Kabushiki Kaisha | Method for the diagnosis of hepatic carcinoma |
| DE3807440A1 (en) * | 1988-03-07 | 1989-09-21 | Progen Biotechnik Gmbh | Method for the immunological detection of substances, and a composition and a test kit |
| US5779976A (en) * | 1988-11-03 | 1998-07-14 | Igen International, Inc. | Apparatus for improved luminescence assays |
| US5798083A (en) * | 1988-11-03 | 1998-08-25 | Igen International, Inc. | Apparatus for improved luminescence assays using particle concentration and chemiluminescence detection |
| US5746974A (en) * | 1988-11-03 | 1998-05-05 | Igen International, Inc. | Apparatus for improved luminescence assays using particle concentration, electrochemical generation of chemiluminescence and chemiluminescence detection |
| US5705402A (en) * | 1988-11-03 | 1998-01-06 | Igen International, Inc. | Method and apparatus for magnetic microparticulate based luminescence assay including plurality of magnets |
| US5962218A (en) * | 1988-11-03 | 1999-10-05 | Igen International Inc. | Methods and apparatus for improved luminescence assays |
| NZ231727A (en) * | 1988-12-12 | 1992-01-29 | Commw Serum Lab Commission | Specific binding type assay (e.g. immunoassay) involving binding compound coupled or adsorbed on inner surface of reaction vessel and lyophilised conjugate |
| US5176999A (en) * | 1989-12-07 | 1993-01-05 | Eastman Kodak Company | Buffered wash composition, insolubilizing composition, test kits and method of use |
| ZA92804B (en) | 1991-02-06 | 1992-12-30 | Igen Inc | Methods and apparatus for improved luminescence assays |
| US6201109B1 (en) | 1993-01-13 | 2001-03-13 | Dade Behring Marburg Gmbh | Assay for bone alkaline phosphatase |
| FR2710410B1 (en) * | 1993-09-20 | 1995-10-20 | Bio Merieux | Method and device for determining an analyte in a sample. |
| EP0745850B1 (en) * | 1994-02-19 | 2005-11-16 | Seikagaku Corporation | Method of assaying normal aglycan, assaying kit, and method of judging joint-related information |
| WO2000013023A1 (en) | 1998-09-01 | 2000-03-09 | Taiho Pharmaceutical Co., Ltd. | Method for assaying human thymidylate synthase and assay kit |
| WO2000062073A1 (en) | 1999-04-12 | 2000-10-19 | Sumitomo Chemical Company, Limited | Method for analyzing the amount of intraabdominal adipose tissue |
| EP1893990B1 (en) | 2005-06-03 | 2018-05-02 | Board of Regents of the University of Texas System | Electrochemistry and electrogenerated chemiluminescence with a single faradaic electrode |
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| GB1572220A (en) * | 1976-10-07 | 1980-07-30 | Mochida Pharm Co Ltd | Immunochemical process of measuring physiologically active substances |
| GB1549069A (en) * | 1976-12-10 | 1979-08-01 | Erba Farmitalia | Enzyme linked immunoassay |
| US4244940A (en) * | 1978-09-05 | 1981-01-13 | Bio-Rad Laboratories, Inc. | Single-incubation two-site immunoassay |
| JPH0235944B2 (en) * | 1980-06-20 | 1990-08-14 | Unilever Nv | TOKUITEKIKETSUGOKENTEIOJITSUSHISURUHOHOOYOBIGAIHOHOOJITSUSHISURUTAMENOSHIKENKITSUTO |
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| DK151399C (en) | 1988-09-05 |
| AT373398B (en) | 1984-01-10 |
| ATA186481A (en) | 1983-05-15 |
| NO159620C (en) | 1989-01-18 |
| NL8101860A (en) | 1981-11-16 |
| IT1137344B (en) | 1986-09-10 |
| SE460930B (en) | 1989-12-04 |
| SE8102558L (en) | 1981-10-26 |
| FR2481318B1 (en) | 1984-10-19 |
| AU542563B2 (en) | 1985-02-28 |
| AU6981181A (en) | 1981-10-29 |
| IT8121122A0 (en) | 1981-04-13 |
| GB2074727B (en) | 1983-11-30 |
| DE3115115C2 (en) | 1987-02-12 |
| FR2481318A1 (en) | 1981-10-30 |
| CA1160566A (en) | 1984-01-17 |
| GB2074727A (en) | 1981-11-04 |
| DE3115115A1 (en) | 1982-02-04 |
| DK155881A (en) | 1981-10-26 |
| NL187545B (en) | 1991-06-03 |
| NO159620B (en) | 1988-10-10 |
| NO811407L (en) | 1981-10-26 |
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