SE460930B - IMMUNOLOGICAL PROCEDURE FOR DETERMINING A SUBSTANCE WHICH ONE OF THE REACTION PARTIES IS BONDED TO A WATER-INSOLABLE CARRIER - Google Patents
IMMUNOLOGICAL PROCEDURE FOR DETERMINING A SUBSTANCE WHICH ONE OF THE REACTION PARTIES IS BONDED TO A WATER-INSOLABLE CARRIERInfo
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- SE460930B SE460930B SE8102558A SE8102558A SE460930B SE 460930 B SE460930 B SE 460930B SE 8102558 A SE8102558 A SE 8102558A SE 8102558 A SE8102558 A SE 8102558A SE 460930 B SE460930 B SE 460930B
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- 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
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- 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
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Description
2 4 6 Û 9 3 ÛSom exempel på testsubstanser kan nämnas alla enskilda beståndsdelar i 10 15 20 25 30 35 fysiologiska vätskor, cell- eller vävnadsextrakt, för vilka immunologiska reak- tionspartner finns eller kan bildas och som har tvâ eller flera immunologiskt aktiva ställen. Därtill hör peptider, proteiner, lipoproteiner, giykoproteiner, steriner, steroider, lipoider, nukleinsyror, enzymer, hormoner, polysackarider, alkaloider. Föredragna immunologiskt aktiva substanser är de naturliga anti- generna såsom hormoner, enzymer, organspecifika antigener, bindvävskomponen- ter, blodcellantigener, plasmaproteiner, patologiska globuliner. Särskilt före- dragna substanser är karcinoembryonalt antigen (CEA) liksom humant choriongo- nadotropin (HCG). vid metoden enligt uppfinningen tjänstgör den immunologiskt aktiva reaktionspartner, som är försedd med ett enzym, som indikator på den immuno- logiska reaktionen. Föredragna enzymer är alkaliskt fosfatas, malat-dehydroge- nas, glukos-s-fosfat-dehydrogenas, glukosoxidas, glukoamylas, galaktosidas och acetylkolinesteras. En särskilt föredragen enzymmarkör är peroxidas från pepparrot. 2 4 6 Û 9 3 ÛAs examples of test substances may be mentioned all individual constituents of physiological fluids, cell or tissue extracts, for which immunological reaction partners exist or can be formed and which have two or more immunologically active sites. These include peptides, proteins, lipoproteins, glycoproteins, sterins, 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 carcinoembryonic antigen (CEA) as well as human chorionic gonadotropin (HCG). in the method according to the invention, the immunologically active reaction partner, which is provided with an enzyme, serves as an indicator of the immunological reaction. Preferred enzymes are alkaline phosphatase, malate dehydrogenase, glucose-s-phosphate dehydrogenase, glucose oxidase, glucoamylase, galactosidase and acetylcholinesterase. A particularly preferred enzyme marker is horseradish peroxidase.
Enzymet som indikator för den immunologiska reaktionen mäts i den flytande, dock företrädesvis i den fasta fasen enligt kända metoder och utgör ett mått på mängden substans som skall bestämmas. Vid peroxidas från pepparrot som markör mäts enzymmängden företrädesvis på grundval av den förhanden- varande katalytiska aktiviteten, vilken bestäms med hjälp av HZOZ och o-fenylendiamin som redoxindikator. Därvid mäts färgintensiteten hos den oxiderade redoxindikatorn fotometriskt efter 30 minuters katalytisk reaktion.The enzyme as an indicator of the immunological reaction is measured in the liquid, but preferably in the solid phase according to known methods and constitutes a measure of the amount of substance to be determined. In the case of peroxidase from horseradish as a marker, the amount of enzyme is preferably measured on the basis of the present catalytic activity, which is determined with the aid of H 2 O 2 and o-phenylenediamine as redox indicator. The color intensity of the oxidized redox indicator is measured photometrically after 30 minutes of catalytic reaction.
Den andra immunologiskt aktiva reaktionspartnern tjänar till separation av den substans som skall bestämmas ur analysprovet. För detta separationssteg kan den andra immunologiskt aktiva reaktionspartnern från början vara bunden till en vattenolöslig bärare eller bindas till en motsvarande bärare under eller efter den immunologiska reaktionen.The other immunologically active reaction partner serves to separate the substance to be determined from the assay sample. For this separation step, the second immunologically active reaction partner may initially be bound to a water-insoluble carrier or bound to a corresponding carrier during or after the immunological reaction.
Lämpliga som vattenolösliga bärare för den andra immunologiskt aktiva reaktionspartnern är: organiska och oorganiska polymerer (amylas, dextran, nativ eller modifierad cellulosa, polyakrylamid, agaros, magnetit, poröst glaspulver, polyvinylidenfluorid (Kynar, framställd av Penn Salt Chemical Corporation, Philadelphia, USA) och latex), innerväggen på testkärl (provrör, titrerplattor eller kyvetter av glas eller syntetmaterial) liksom ytan på fasta kroppar (glas- och syntetmaterialstavar, stavar med ändförtjockriing, stavar med ändvingar eller -lameller). Särskilt lämpliga bärare för metoden enligt uppfinningen är glas- och syntetmaterialkulor.Suitable water-insoluble carriers for the other immunologically active reactant are: organic and inorganic polymers (amylase, dextran, native or modified cellulose, polyacrylamide, agarose, magnetite, porous glass powder, polyvinylidene fluoride (Kynar, manufactured by Penn Salt Chemical Corporation, Philadelphia). and latex), the inner wall of test vessels (test tubes, titrating plates or cuvettes of glass or synthetic material) as well as the surface of solid bodies (glass and synthetic material rods, end-thickening rods, end-wing or lamella rods). Particularly suitable carriers for the method according to the invention are glass and synthetic material beads.
Den andra immunologiskt aktiva reaktionspartriern kan vara fysikaliskt (adsorptivt) eller kemiskt bunden till den vattenolösiiga bäraren eller bindas med ._ ...e ...___...-....._........~,....,. . l0 15 20 25 30 35' 3 hjälp av en ytterligare reaktionspartner, som i sin tur är bunden tifi ênqaärgrš, under eller efter reaktionen.The second immunologically active reactant may be physically (adsorptively) or chemically bound to the water-insoluble carrier or bound with ._ ... e ...___...-....._........ ~ , ....,. . by means of an additional reaction partner, which in turn is bound to fi ênqaärgrš, during or after the reaction.
Väsentligt för metoden enligt uppfinningen är att den substans som skall bestämmas har minst tvâ immunologislrt aktiva ställen (epitoper), som kan kännas igen och bringas till reaktion av de bada immunologiskt aktiva reaktionspart- nerna, den till en bärare bundna och den med ett enzym försedda reaktionspart- nern. Som immunologiskt aktiva reaktionspartner utnyttjas tva olika monoklona- la antikroppar eller en monoklonal antikropp och en antikropp från en annan djurart, varvid de båda kombinationerna reagerar med den substans som skall bestämmas, men som vardera är riktade mot olika immunologiskt aktiva ställen.It is essential for the method according to the invention that the substance to be determined has at least two immunologically active sites (epitopes), which can be recognized and reacted by the two immunologically active reactants, the one bound to a carrier and the one provided with an enzyme. the reaction partner. As immunologically active reactants, two different monoclonal antibodies or a monoclonal antibody and an antibody from another animal species are used, the two combinations reacting with the substance to be determined, but which are each directed to different immunologically active sites.
För bestämning av antigener är kombinationen av antikroppar av olika kloner eller av monoklonala antikroppar och antikroppar från en annan djurart särskilt lämplig.For the determination of antigens, the combination of antibodies of different clones or of monoclonal antibodies and antibodies of another animal species is particularly suitable.
För den immunologiska reaktionen kan provet användas med den substans, som skall bestämmas, antingen direkt eller förutspätt eller förbe- handlat på något lämpligt sätt. För förbehandling kan den substans som skall bestämmas isoleras, anrikas eller befrias från störande beståndsdelar.For the immunological reaction, the sample can be used with the substance to be determined, either directly or pre-diluted or pretreated in any suitable way. For pre-treatment, the substance to be determined can be isolated, enriched or freed from disturbing constituents.
Enligt förfarandet enligt uppfinningen bringas den substans som skall bestämmas samtidigt till reaktion med den enzymmärkta och den bärarbundna immunologiskt aktiva reaktionsparmern. Ordningsföljden på reagenstillsatsen beror pà karaktären på det valda bärarsystemet. När man arbetar med en sensibiliserad plastkula sammanförs först den erizymmärkta reaktíonsparmem med den substans som skall bestämmas i nagot lämpligt provrör, och därefter tillsätts den med den andra reaktionspartriern sensibiliserade kulan.According to the process of the invention, the substance to be determined is simultaneously reacted with the enzyme-labeled and the carrier-bound immunologically active reaction parent. The order of the reagent additive depends on the nature of the selected carrier system. When working with a sensitized plastic bead, first combine the erizzyme-labeled reaction sperm with the substance to be determined in some suitable test tube, and then add the bead sensitized with the other reaction batch.
Den immunologiska reaktionen genomförs i nagot lämpligt buffertsys- tem för att hälla ett optimalt pH-värde, som kan ligga mellan ß och 9.The immunological reaction is carried out in a suitable buffer system to obtain an optimal pH value, which can be between ß and 9.
Föredragna buffertar är exempelvis acetatbuffert, citratbuffert, fosfatbuffert, tris-buffert, trietanolaminbuffert, boratbuffert eller glycinbuffert. Man kan också använda buffertblandningar.Preferred buffers are, for example, acetate buffer, citrate buffer, phosphate buffer, tris buffer, triethanolamine buffer, borate buffer or glycine buffer. You can also use buffer mixtures.
Den immunologiska reaktionen sker företrädesvis vid en temperatur mellan 0 och 55°C. Normalt ökar den immunologiska reaktionshastigheten med högre temperatur, varvid jämvikten uppnås snabbare under i övrigt lika testbetingelser. lnkubationen av den substans, som skall bestämmas, med den enzym- märkta reaktionspartnern och den bärarbundna reaktionspartnern kan ske tills man uppnår jämvikt. Den immunologiska reaktionen kan dock också stoppas vid en tidigare tidpunkt genom att efter en bestämd inkubationstid de fasta och flytande faserna separeras och graden av enzymmärkning bestäms antingen i den flytande elleri den fasta fasen. 460 930 ' 4 10 15 20 25 30 35' Vid den immunologiska reaktionen kan man vidta åtgärder för stabilise- ring av den immunologiska aktiviteten hos reaktionspartnerna och den substans som skall bestämmas liksom av enzymet. Vidare kan man till inkubationsiös- ningen tillsätta beståndsdelar, såsom proteiner och detergenter, för att utesluta ospecifika reaktioner, för att minska hämmande påverkan eller för aktivering.The immunological reaction preferably takes place at a temperature between 0 and 55 ° C. Normally, the immunological reaction rate increases with higher temperature, whereby the equilibrium is reached more quickly under otherwise equal test conditions. 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 stopped at an earlier time point by separating the solid and liquid phases after a certain incubation period and the degree of enzyme labeling is determined either in the liquid or in the solid phase. 460 930 '4 10 15 20 25 30 35' In 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 by the enzyme. Furthermore, constituents, such as proteins and detergents, can be added to the incubation solution to exclude non-specific reactions, to reduce inhibitory effects or for activation.
Den nya metoden enligt föreliggande uppfinning är utomordentligt känslig och utmärks av sin enkelheti handhavandet.The new method of the present invention is extremely sensitive and is characterized by its simplicity of operation.
Uppfinningen illustreras i de efterföljande exemplen.The invention is illustrated in the following examples.
Exemæi i Kvantitativ bestämning av CEA i patientplasma med en monoklonal CEA-anti- kropp och en sedvanlig CEA-antikropïgtl l erforderligt antal provrör ( 10 x 75 mm) pipetteras i vart och ett 0,2 ml testlösning (0,2 moi/l NaH2P04/Na2HPO4, pH 6,5 med 2 g/l oxserumalbumin, 2096 normalt getserum och 0,2 pg/mi get-anti-CEA-peroxidas-konjugat), 0,050 ml av det patientplasma som skall analyseras respektive GEA-standarder (0 ng/ml CEA, 2,5 ng/mi csA, io ng/mi GEA och zo ng/mi cEA) och cars-kontrollsam- met (5,0 ng/ml CEA i 1,0 ng/ml) tillblandas, en med monoklonalt mus-anti-CEA- sensibiliserad polystyrenkula* (ß = 6,5 mm) tillsätts och inkuberas i 16 timmar vid 37°C. Därefter tvättas polystyrenkuian tre gånger med vardera 2-5 ml dest. vatten, överförs till vardera 0,5 ml substratbuffert för peroxidasaktivitets- benämningen (0,1 moi/l kaliumcitratbuffert av pH 5,0 med 6 mmol/l HZOZ och 20 mmol/l o-fenylendiamin) och inkuberas under 30 minuter vid rumstemperatur (22°C). För att stoppa den peroxidatiska aktiviteten liksom för att intensifiera färgintensiteten tillblandas 2,0 ml lN HCi, odi man mäter inom 30 minuter extinktionen vid våglängden 092 nm fotometriskt. I Tabell i anges värdena för en CEA-bestämning och jämförs med de värden, som erhölls med Radioimmunoassay från ROCHE.Examples of Quantitative determination of CEA in patient plasma with a CEA monoclonal antibody and a conventional CEA antibody required number of test tubes (10 x 75 mm) are pipetted into each 0.2 ml test solution (0.2 mol / l NaH 2 PO 4 / Na2HPO4, pH 6.5 with 2 g / l bovine serum albumin, 2096 normal goat serum and 0.2 pg / ml goat anti-CEA peroxidase conjugate), 0.050 ml of the patient plasma to be analyzed and GEA standards (0 ng / ml CEA, 2.5 ng / ml csA, io ng / ml GEA and zo ng / ml cEA) and the cars control kit (5.0 ng / ml CEA at 1.0 ng / ml) are mixed, one with mouse monoclonal anti-CEA sensitized polystyrene bead * (ß = 6.5 mm) is added and incubated for 16 hours at 37 ° C. Then the polystyrene well is washed three times with 2-5 ml of dist each. water, transferred to each 0.5 ml of substrate buffer for the peroxidase activity label (0.1 mol / l potassium citrate buffer of pH 5.0 with 6 mmol / l H 2 O 2 and 20 mmol / l o-phenylenediamine) and incubated for 30 minutes at room temperature ( 22 ° C). To stop the peroxidic activity as well as to intensify the color intensity, 2.0 ml of 1N HCl are added, and the extinction at 092 nm is measured photometrically within 30 minutes. Table i lists the values for a CEA assay and compares them with the values obtained by Radioimmunoassay from ROCHE.
*I Framställningen av monoklonalt mus-anti-CEA sker analogt med den i Journal of lmmunologicai Methods, 32 (1980) 297-304, beskrivna metoden, varvid som utgàngscellinje för fusionen myelomlinjen Sp 2/0l-AG används, vilken deponerats vid ATCC under nr. CRL 8006. Fusionen sker med mjältceller från möss, som immuniserats med CEA. immuniseringen av mössen utfördes analogt med Tabell I i nämnda publikation, varvid de första båda immuniseringarna skedde med vardera 50 pg CEA, lmmuniseringarna 3 och i; utelämnades, immuniseringen 5 utfördes med 50 pg CEA och immuniseringarna 6-8 utfördes med vardera 200 pg CEA.* In the preparation of mouse anti-CEA monoclonal, the method is carried out analogously to the method described in the Journal of Immunological Methods, 32 (1980) 297-304, using as my starting cell line for the fusion the myeloma line Sp 2 / O1-AG, which was deposited with the ATCC during no. CRL 8006. The fusion takes place with spleen cells from mice, which have been immunized with CEA. the immunization of the mice was performed analogously to Table I in said publication, the first two immunizations taking place with 50 pg CEA each, the immunizations 3 and i; were omitted, immunization 5 was performed with 50 pg CEA and immunizations 6-8 were performed with 200 pg CEA each.
Tabell I 460 930 Testmaterial ^E492 nm/RT/30 min.Table I 460 930 Test material ^ E492 nm / RT / 30 min.
CEA-Standard 0 ng/ml CEA 2,5 ng/ml CEA 10,0 ng/ml CEA 20,0 ng/ml CEA 0,103 0,330 0,978 1,850 CEÅ- kontrollserum 5,0 ng/ml CBA 0,540 Patientglasma Nr. 7212 Nr. 7188 Nr. 7220 Nr. 7218 Nr. 7234 Nr. 7249 Nr. 7203 Nr. 7223 Nr. 7247 Nr. 7258 Nr. 7215 Nr. 7219 Nr. 8180 Nr. 7248 Nr. 7262 Nr. 7201 w ~ ~ à ß h) W N N N r-l I-l N O ~ _ _ \O Gi H O k) à UI N N N 0'\ ~ ~ ROCHE-RIA-Test ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml Föríarandet enligt uggf inningen 1,2 ng/ml 1,0 ng/ml 1,4 ng/ml 1,3 ng/ml 2,7 ng/ml 2,0 ng/ml 2,0 ng/ml 3,3 ng/ml 2 ,8 ng/ml 4,3 ng/ml 5,5 ng/ml 5,9 ng/ml 8,5 ng/ml 10,7 ng/ml 14,3 ng/ml 15,3 ng/ml Värden under 2,5 ng/m! CEA ligger i normalområdet, medan värden över 2,5 nglml ligger i det patologiska omrâdet. 460 936- lO 15 20 25 a s Exempel 2 Kvantitativ bestämning av CEA i patientplasma med monoklonala GEA-anti- kgpar från tvâ olika kloner I erforderligt antal provrör (l0 x 75 mm) pipetteras vardera 0,2 ml testlösning (0,2 mol/l NaH2PO4/Na2HPOq, pH 6,5 med 2 g/l oxserumalbumin, 2096 normalt getserum och 0,15 tig/ml monoklonalt mus-anti-CEA-peroxidas- konjugat* ), 0,050 ml av det patientplasma som skall analyseras respektive av GEA-standard (O ng/ml CEA, 2,5 ng/ml CEA, 10 ng/ml CEA och 20 ng/ml CEA), och CEA-kontrollserumet (5,0 ng/ml CEA i 1,0 ng/ml) tillblandas, en med monoklonalt mus-anti-CEA sensíbiliserad x polystyrenkula (0 = 6,5 mm) tillsätts och man inkuberar i l6 timmar vid 37°C. Därefter tvättas polystyrenkulorna tre gånger med vardera 2-5 ml dest. vatten, överförs till vardera 0,5 ml substratbuffert för peroxidasaktivitetsbestämningen (0,1 mol/l kaliumcitrat- buffert av pH 5,0 med 6 mmol/l H202 och 20 mmol/l o-fenylendiamin) och inkuberas under 30 minuter vid rumstemperatur (22°C). För att stoppa den peroxidatiska aktiviteten liksom för att intensifiera färgintensiteten tillblandas 2,0 ml lN HCl och inom loppet av 30 minuter mäts extinktionen vid våglängden 492 nm fotometriskt. l Tabell lll är värdena för en GEA-bestämning angivna och jämförs med värden erhållna med Radioimmunoassay från ROCHE.CEA Standard 0 ng / ml CEA 2.5 ng / ml CEA 10.0 ng / ml CEA 20.0 ng / ml CEA 0.103 0.330 0.978 1.850 CEÅ control serum 5.0 ng / ml CBA 0.540 Patient glass no. 7212 Nr. 7188 Nr. 7220 Nr. 7218 Nr. 7234 Nr. 7249 Nr. 7203 Nr. 7223 Nr. 7247 Nr. 7258 Nr. 7215 Nr. 7219 Nr. 8180 Nr. 7248 Nr. 7262 Nr. 7201 w ~ ~ à ß h) WNNN rl Il NO ~ _ _ \ O Gi HO k) à UI NNN 0 '\ ~ ~ ROCHE-RIA-Test ng / ml ng / ml ng / ml ng / ml ng / ml ng / ml ng / ml ng / ml ng / ml ng / ml ng / ml ng / ml ng / ml ng / ml ng / ml ng / ml The procedure according to the invention is 1.2 ng / ml 1.0 ng / ml 1, 4 ng / ml 1.3 ng / ml 2.7 ng / ml 2.0 ng / ml 2.0 ng / ml 3.3 ng / ml 2.8 ng / ml 4.3 ng / ml 5.5 ng / ml 5.9 ng / ml 8.5 ng / ml 10.7 ng / ml 14.3 ng / ml 15.3 ng / ml Values below 2.5 ng / m! CEA is in the normal range, while values above 2.5 nglml are in the pathological range. Example 2 Quantitative determination of CEA in patient plasma with GEA monoclonal anti-kg pairs from two different clones In a required number of test tubes (10 x 75 mm) each pipette 0.2 ml test solution (0.2 mol / 1 NaH2PO4 / Na2HPOq, pH 6.5 with 2 g / l bovine serum albumin, 2096 normal goat serum and 0.15 tig / ml mouse anti-CEA peroxidase conjugate *), 0.050 ml of the patient plasma to be analyzed and of GEA respectively standard (0 ng / ml CEA, 2.5 ng / ml CEA, 10 ng / ml CEA and 20 ng / ml CEA), and the CEA control serum (5.0 ng / ml CEA in 1.0 ng / ml) are mixed, a mouse poly-anti-CEA monoclonal monoclonal x polystyrene bead (0 = 6.5 mm) is added and incubated for 16 hours at 37 ° C. The polystyrene beads are then washed three times with 2-5 ml of dist each. water, each is transferred to 0.5 ml of substrate buffer for the peroxidase activity assay (0.1 mol / l potassium citrate buffer of pH 5.0 with 6 mmol / l H 2 O 2 and 20 mmol / l o-phenylenediamine) and incubated for 30 minutes at room temperature ( 22 ° C). To stop the peroxidic activity as well as to intensify the color intensity, 2.0 ml of 1N HCl are added and within 30 minutes the extinction at the wavelength of 492 nm is measured photometrically. In Table III, the values for a GEA assay are given and compared with values obtained by Radioimmunoassay from ROCHE.
*Framställningen av monoklonalt mus-anti-CEA sker analogt med den i Journal of Immunological Methods, 32 (1980) 297-300, beskrivna metoden, varvid som utgångscellirlje för fusionen används myelomlinjen Sp 2/0l-Ag, som deponerats vid ATCC under nr. CRL 8006. Fusionen sker med mjältcelier från möss, som immuniserats med CEA. Musimmuniseringen skedde analogt med Tabell l i den angivna publikationen, varvid de första båda immuniseringarna utfördes med vardera 50 ug CEA, immuniseringarna 3 och b utelämnades, immunisering 5 utfördes med 50 ug CEA och immuniseringarna 6-8 med vardera 200 ug CEA. Man använder tvâ olika, lämpliga monoklonala antikroppar, som är riktade mot olika epitoper hos CEA-antigenet. 7 Tabell II 460 930 Testmaterial A 5492 nm/nr/ao CBA~Standard 0 ng/ml CEA 2,5 ng/ml CEA 10,0 ng/ml CEA 20,0 ng/ml CEA 0,390 o,44o 0,620 o,aeo CEA-kontrollserum 5,0 ng/ml CEA 0,510 Paflengaflna N;_ 7220 N;_ 7234 Nr, 7223 Nr_ 7247 Nr, 7258 Nr, 7215 Nr_ 7219 Nr. 8180 Nr. 7248 Nr. 7262 Nr. 7201 ROCHE-RIA-Test 1,2 ng/ml 2,5 ng/ml 3,0 ng/ml 3,1 ng/ml 4,6 ng/ml 4,9 ng/ml 5,0 ng/ml _8,6 ng/ml 11,2 ng/ml 14,2 ng/ml 15,6 ng/ml Förfarandet enligt uggfinningen o,e 3,0 3,5 3,2 4,4 s,o 5,4 8,6 11,0 14.0 16,0 ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml Värden under 2,5 ng/ ml CEA ligger i normalomrâdet, medan värden över 2,5 ng/ml ligger i det patologiska området. 460 936 10 15 20 25 8 Exempel 3 Kvantitativ bestämning av HCG i serum med monoklonala HCG-antikroppar från tvâ olika kloner l erforderligt antal provrör (10 x 75 mm) pipetteras vardera 0,2 ml testlösning (0,1 moi/l NaHzPOalNazHPOk, pH 7,0 med 2 g/l oxserumalbumin och 1,0 gig/ml monoklonalt mus-anti-HCG-peroxidas-konjugatx), 0,050 ml av det patientplasma som skall analyseras respektive HCG-standarder (0, 25, 50, 100 och 200 miU/ml HCG) tillblandas, vardera en med monoklonalt mus-anti-HCG senszbafisefad ponynyfenkuia* (o = 6,5 mm) means och :nkuberas tex. vid a7°c under tvâ timmar. Därefter tvättas polystyrenkulorna tre gånger med vardera 2-5 mi dest. vatten, överförs till vardera 0,5 ml substratbuffert för aktivitets- bestämning av peroxidasen (0,1 mol/ml kaliumcitratbuffert av pH 5,0 med 6 mmolll 11202 och 20 mmol/l o-fenylendiamin) och inkuberas under 30 minuter vid rumstemperatur (l6-30°C). För att stoppa den peroxidatiska aktiviteten liksom för att intensifiera färgintensiteten tillblandas 1,0 ml 2N HCl och inom 30 minuter mäts extinktionen vid våglängden 1492 nm fotometriskt. Itabellen anges värden för HCG-bestämningar i serum.* The production of mouse anti-CEA monoclonal is analogous to the method described in Journal of Immunological Methods, 32 (1980) 297-300, using as my starting cell line for the fusion the myeloma line Sp 2/01-Ag, which was deposited with the ATCC under no. . CRL 8006. The fusion takes place with spleen cells from mice, which have been immunized with CEA. The mouse immunization was performed analogously to Table 1 in the cited publication, with the first two immunizations performed with 50 μg CEA each, the immunizations 3 and b omitted, immunization 5 performed with 50 μg CEA and the immunizations 6-8 with 200 μg CEA each. Two different, suitable monoclonal antibodies are used, which target different epitopes of the CEA antigen. 7 Table II 460 930 Test material A 5492 nm / nr / ao CBA ~ Standard 0 ng / ml CEA 2.5 ng / ml CEA 10.0 ng / ml CEA 20.0 ng / ml CEA 0.390 o, 44o 0.620 o, aeo CEA control serum 5.0 ng / ml CEA 0.510 Pa fl enga fl na N; _ 7220 N; _ 7234 Nr, 7223 Nr_ 7247 Nr, 7258 Nr, 7215 Nr_ 7219 Nr. 8180 Nr. 7248 Nr. 7262 Nr. 7201 ROCHE-RIA-Test 1.2 ng / ml 2.5 ng / ml 3.0 ng / ml 3.1 ng / ml 4.6 ng / ml 4.9 ng / ml 5.0 ng / ml _8, 6 ng / ml 11.2 ng / ml 14.2 ng / ml 15.6 ng / ml The procedure according to the invention o, e 3.0 3.5 3.2 4.4 s, o 5.4 8.6 11 .0 14.0 16.0 ng / ml ng / ml ng / ml ng / ml ng / ml ng / ml ng / ml ng / ml ng / ml ng / ml ng / ml Values below 2.5 ng / ml CEA are in normal range, while values above 2.5 ng / ml are in the pathological range. 460 936 10 15 20 25 8 Example 3 Quantitative determination of HCG in serum with HCG monoclonal antibodies from two different clones In the required number of test tubes (10 x 75 mm) each pipette 0.2 ml test solution (0.1 moi / l NaHzPOalNazHPO pH 7.0 with 2 g / l ox serum albumin and 1.0 g / ml mouse-anti-HCG peroxidase conjugatex), 0.050 ml of the patient plasma to be analyzed and HCG standards (0, 25, 50, 100 and 200 ml / ml HCG) is mixed, each one with monoclonal mouse anti-HCG sensitized ponynyfenkuia * (o = 6.5 mm) means and: incubated e.g. at a7 ° c for two hours. Then the polystyrene beads are washed three times with 2-5 ml each. water, each is transferred to 0.5 ml of substrate buffer to determine the activity of the peroxidase (0.1 mol / ml potassium citrate buffer of pH 5.0 with 6 mmol / l 11202 and 20 mmol / l of o-phenylenediamine) and incubated for 30 minutes at room temperature ( 16-30 ° C). To stop the peroxidic activity as well as to intensify the color intensity, 1.0 ml of 2N HCl is added and within 30 minutes the extinction at the wavelength of 1492 nm is measured photometrically. The table lists values for HCG determinations in serum.
*Franiställningen av de monoklonala mus-HCG-antikropparna sker enligt den i Journal of lmmunological Methods, 32 (1980) 297-300 beskrivna metoden. Man använder tvâ olika, lämpliga monoklonala antikroppar, som är riktade mot olika epitoper hos HCG-antigenet.* The release of the mouse HCG monoclonal antibodies is according to the method described in Journal of Immunological Methods, 32 (1980) 297-300. Two different, suitable monoclonal antibodies are used, which target different epitopes of the HCG antigen.
HCG-bestämningfinumanserum (medelvärde av dubbelbestämningar) 1. Standardkurvor AE 492 nm/30 Mirn/RT.HCG determination fi numerical range (mean of double determinations) 1. Standard curves AE 492 nm / 30 Mirn / RT.
Tu IU/ml HCG 1) Serum ' 2) Plasma 3) Urin 4) Buffert 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 HCG-värdena för de nedan angivna exemplen på patientserum avlästes ur standardkurvan l). Standardkurvorna 2), 3) och 4) bestämdes en annan dag med nya HCG-standarder.Tu IU / 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 The HCG values for the examples of patient serum given below were read from the standard curve l). The standard curves 2), 3) and 4) were determined another day with new HCG standards.
D» r" *77e-T7«.»..1e..~.-.-.-...1.4.,.1 9 460 930 2. Patientsera ' 82492/30 Min/RT alu/ml acc Serum uppmätt ur kurva l) Peel 091080 0.041 0 Nr. 2801 0.025 ' 0 Nr. 3881 0.450 47 Nr. 2673 1,13 127 Nr. 1167 2.12 (1=2) 470 Nr. 4891 0.975 (1=so) s'4so Nr. 3240 1.06 (1=2o) 2'28o Nr. 4418 1.475 (1:1000) 1s7'o00 10 15 20 Exemæl 16 För sensibilisering av en tlterplatta pipetteras per hålighet 0,15 ml sensibiliseringslösning (50 mmol/l NaHCO3, pH 8,0-8,3 med 20 mg/l avidin), och plattan får stå i en fuktig kammare vid l8-26°C under 10-214 timmar.D »r" * 77e-T7 «.» .. 1e .. ~.-.-.-... 1.4.,. 1 9 460 930 2. Patientsera '82492/30 Min / RT alu / ml acc Serum uppmätt ur curve l) Peel 091080 0.041 0 No. 2801 0.025 '0 No. 3881 0.450 47 No. 2673 1.13 127 No. 1167 2.12 (1 = 2) 470 No. 4891 0.975 (1 = so) s'4so Nr. 3240 1.06 (1 = 2o) 2'28o No. 4418 1.475 (1: 1000) 1s7'o00 10 15 20 Example 16 To sensitize a filter plate, pipette 0.15 ml of sensitization solution (50 mmol / l NaHCO3, pH 8) per cavity. 0-8.3 with 20 mg / l avidin), and the plate is allowed to stand in a humid chamber at 18-26 ° C for 10-214 hours.
Efter utskakning av sensibiliseringslösningen och tvättning 2-3 gånger med PBS pipetteras 0,2 ml buffert per hålighet, plattan försluts med "själv- häftande folie" och förvaras minst l dag vid 2-8°C i en fuktig kammare.After shaking out the sensitization solution and washing 2-3 times with PBS, 0.2 ml of buffer per cavity is pipetted, the plate is sealed with "self-adhesive foil" and stored for at least 1 day at 2-8 ° C in a humid chamber.
Omedelbart före utförandet av CEA-enzymimmunoassay utskakas bufferten. l var och en av de för testserien nödvändiga håligheterna i titerplattan pipetteras 0,1 ml anti-CEA-peroxidas-testlösning, till vilken satts 2 ml/l biotiny- lerat anti-CEA från cellinjen C-l9, samt 0,05 ml CEA-standardlösrtingar resp. värmebehandlade (56°C/ 30 min.) serum- eller plasmaprover. Plattan försluts och inkuaefa; under a timma.- vid 37°C (vattenbad). Efter tvättning av plattan 3-5 gånger med PBS - 0,5 ml/l Twee 20 tillsätts 0,15 ml o-fenylendlamin- substratbuffert per hålighet för bestämning av den bundna mängden peroxidas.Immediately before performing the CEA enzyme immunoassay, the buffer is shaken out. In each of the cavities required for the test series in the titer plate, pipette 0.1 ml of anti-CEA peroxidase test solution, to which 2 ml / l of biotinylated anti-CEA from the C-19 cell line, and 0.05 ml of CEA have been added. standard defaults resp. heat-treated (56 ° C / 30 min.) serum or plasma samples. The plate is sealed and inkuaefa; for one hour at 37 ° C (water bath). After washing the plate 3-5 times with PBS - 0.5 ml / l Twee 20, add 0.15 ml of o-phenylendlamine substrate buffer per cavity to determine the amount of peroxidase bound.
Efter en 30 minuters inkubation vid l8-26°C stoppas den enzymatiska reaktionen genom tillblandning av 0,15 ml 0,5 mol/l 112504 - 2 g/l NazSzOs, och färgintensi- teten vid våglängden 492 nm mäts med flerkanalsfotometer.After a 30 minute incubation at 18-26 ° C, the enzymatic reaction is stopped by mixing 0.15 ml of 0.5 mol / l 112504 - 2 g / l Na 2 S 2 O 3, and the color intensity at the wavelength of 492 nm is measured with a multi-channel photometer.
Exemggl 5 Man arbetar på analogt sätt som i Exempel b, varvid dock titerplattorna är sensibiliserade med biotin och varvid man arbetar med avidinhaltigt monoklonalt anti-GEA.Example 5 The procedure is analogous to that of Example 2, except that the titer plates are biotin sensitized and the avidin-containing anti-GEA monoclonal agent is used.
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| Application Number | Priority Date | Filing Date | Title |
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| CH320980A CH642458A5 (en) | 1980-04-25 | 1980-04-25 | Immunological method |
| CH589880A CH651396A5 (en) | 1980-08-04 | 1980-08-04 | Immunological method for detecting and determining carcinoembryonic antigen |
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| SE460930B true SE460930B (en) | 1989-12-04 |
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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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| AT373398B (en) | 1984-01-10 |
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| NL8101860A (en) | 1981-11-16 |
| IT1137344B (en) | 1986-09-10 |
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| 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 |
| DK151399B (en) | 1987-11-30 |
| NO159620B (en) | 1988-10-10 |
| NO811407L (en) | 1981-10-26 |
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