CA1160566A - Immunological determination method - Google Patents
Immunological determination methodInfo
- Publication number
- CA1160566A CA1160566A CA000373607A CA373607A CA1160566A CA 1160566 A CA1160566 A CA 1160566A CA 000373607 A CA000373607 A CA 000373607A CA 373607 A CA373607 A CA 373607A CA 1160566 A CA1160566 A CA 1160566A
- Authority
- CA
- Canada
- Prior art keywords
- cea
- immunologically active
- process according
- enzyme
- active reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
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
Abstract
ABSTRACT
There is disclosed an enzyme immunoassay according to the so called "sandwich-principle". The essential feature of the novel assay consisto in the fact that not 2 immunological incubations are performed but that the substance to be determined is simultaneously incubated with both immunologically active reaction partners.
There is disclosed an enzyme immunoassay according to the so called "sandwich-principle". The essential feature of the novel assay consisto in the fact that not 2 immunological incubations are performed but that the substance to be determined is simultaneously incubated with both immunologically active reaction partners.
Description
1160~6 The present invention is concerned with an enzyme--immune process 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 reacted with two immunologically active reaction partners. Usually, one of these immunologically active reaction partners is bound to a water-insoluble carrier, while the other is marked with a suitable enzyme. In practice, the substance to be determined is firstly reacted with the reaction partner bound to the carrier, whereupon, after phase separation and washing, the substance which is meanwhile immunologically bound to the carrier is reacted with the second, enzyme-marked reaction partner. After renewed phase separation, the enzymatic reaction for the quantitative detection of the substance to be determined is carried out either in the solid or the liquid phase.
It has hitherto been the opinion that, in the case of enzyme-immune processes, the immunological reaction of the substance to be determined had to be effected with the two corresponding immunologically active reaction partners successively in a first and in a second reaction step.
In the scope of the present invention it has now surprisingly been found that, contrary to the hitherto existing opinion, it is possible to work in e~fect in a "one-pot process", with the substance to be determined being reacted simultaneously with both immunologically active reaction partners during a single incubation.
The present invention is accordingly concerned with an enzyme-immune process for the detection and determination of a substance with an immunologically active reaction partner, which is marked with an enzyme, as well as an immunologically active reaction partner, which is or will be bound to a water-insoluble carrier, by incubation of the substance to be determined with the immunologically active reaction partners, subsequent separation of solid and liquid phases and measurement of the extent of the enzyme marking either in the solid or in the liquid phase as the extent of the amount of the substance to be determined, which is characterised in that for the immunological reaction the substance to be determined as well as the immunologically active reaction partners are incubated from the outset and together and only once.
The substance to be determined can be any constituents in physiological fluids, cell extracts or tissue extracts for which there are present or can be formed immunological reaction partners, and which possess two or more immuno-logically active sites. Such substances include peptides, ~l~O';~
proteins, lipoproteins, glycoproteins, sterols, steroids, lipoids, nucleic acids, enzymes, ho:rmones, polysaccharides and alkaloids, Preferred immunologically active substances are natural antigens such as hormones, enzymes, organ--specific antigens, connective tissue components, blood cell antigens, plasma proteins and pathological globulins.
Especially preferred substances are carcinoembryonic antigen (CEA) as well as human choriongonadotropin (HCG).
Thus the present invention provides an immunoassay process for determining substances having at least two immuno-logically active sites comprising:
a) preparing a mixture of (i) a substance having at least two immunologically active sites or epitopes, (ii) an immunologically active reaction partner having an enzyme marker, and (iii) an immunologically active reaction partner which is or will be bound to a water insoluble carrier, b) incubating said mixture, c) separating the solid and liquid phase, d) determining the amount of substance by measuring the extent of the enzyme marking in the solid or liquid phase, the improvement consisting in e) using different monoclonal antibodies as the immunologi-cally active reaction partners, orf) using a monoclonal antibody and an antibody from a dif-ferent animal species as the immunologically active ~-~ reaction partners.
116~S~j - 3a -In the method in accordance with the invention the immunologically active reaction partner, which is marked with an enzyme, serves as the indicator of the immunological reac-tion. Preferred enzymes are alkaline phosphatase (EC 3.1.3.1), malate dehydrogenase (EC 1.11.37), glucose-6-phosphate de-hydrogenase (EC 1.1.1.49), glucose oxidase (EC 1.1.3.4), gluco-amylase (EC 3.2.1.3), galactosldase (EC 3.2.1.22/3.2.1.23) and acetylcholine esterase (EC 3.1.1.7). Peroxidase from horse-radish (EC 1.11.1.7) is an especially preferred enzyme label.
The enzyme as the indicator of the immunological reaction is measured in the liquid phase or, preferably, in the solid phase according to known methods and is a measurement for the amount of the substance to be determined. When the label is peroxidase from horseradish the amount of enzyme is preferably measured on the basis of the catalytic activity present, which is determined with the aid of hydrogen peroxide and o-phenylenediamine as the redox indicator. In ., ,6 this case, after a catalytic reaction lasting for 30 minutes the colour intensity of the oxid:Lsed redox indicator is measured photometrically.
The second immunologically active reaction partner serves for ~he separation of the substance to be determined from the analytical sample. For this separation step the second immunologically active reaction partner can be bound to a water-insoluble carrier from the outset or can be bound to such a carrier during or after the immunological reaction.
Suitable water-insoluble carriers for the second immunologicaliy active reaction partner are organic and inorganic polymers [amylases, dextrans, natural or modified celluloses, polyacrylamides, agaroses, magnetite, porous glass powder, polyvinylidene fluoride (Kynar) and latex], the inner wall of test vessels (test tubes, titre plates or cuvettes of glass or artificial materials) as well as the surface of solid bodies (rods of glass and artificial material, rods with terminal thickening, rods with terminal lobes or lamellae). Spheres of glass and artificial material are especially suitable carriers for the method in accordance with the invention.
~ he second immunologically active reaction partner can be bound to the water-insoluble carrier physically (a~sorptively) or chemically or with the aid of a further reaction partner, which, in turn, is bound to a carrier.
It is essential for the method in accordance with the invention that the substance to be determined possesses at least two immunologically active sites (epitopes), which can be recognised by and react with the two immunologically active reaction partners, the reaction partner which is bound to a carrier and the reaction partner which is marked with an enzyme. As the immunologically active reaction partners there are preferably used two different components, which indeed both react with the substance to be determined, but each cf which is directed to different immunologically active sites. For the determinatlon of antigens there are especially suitable two antibodies for the particular antigen, the antibodies having been produced in two different animal species and being directed towards different epitopes of the antigen. The combination of antibodies of different - clones or of monoclonal antibodies and antibodies from a different animal species is particularly suitable.
For the immunological reaction the sample with the substance to be determined can be used directly or can be pre-diluted or pre-treated in a suitable manner. For the pre-treatment the substance to be determined can be isolated, enriched or freed from troublesome ingredients.
According to the process in accordance with the invention, the substance to be determined is rea~ted simultaneously with the enzyme-marked as well as the carrier--bound immunologically active reaction partner. The sequence in which the reagents are added is governed by the type of carrier system chosen. When a sensitised plastic sphere is used, the enzyme-marked reaction partner and the substance to be determined are initially placed together in a suitable test tube and subsequently the sphere sensitised with the other reaction partner is added.
The immunological reaction is carried out in a suitable buffer system so as to maintain an optimum pH-value, which can lie between 4 and 9. Preferred buffers are, for example, acetate buffer, citrate buffer, phosphate buffer, tris buffer, triethanolamine buffer, borate buffer or glycine buffer. Buffer mixtures can also be used.
The immunological reaction is preferably carried out at a temperature between 0C and 55C. Normally, the immunological reaction velocity increases with higher temperatures, whereby under otherwise similar test conditions the equilibrium is achieved more rapidly.
The incubation of the substance to be determined with the enzyme-marked reaction partner and the carrier-bound reaction partner can be carried out until the equilibrium - 25 has been reached. The immunological reaction can, however, 'iÇ;6 also be stopped at an earlier point in time by separating the solid and the liquid phase after a defined incubation period and measuring the extent of the enzyme marking either in the liquid phase or in the solid phase.
In the immunological reaction precautions can be taken to stabilize the immunological activity of the reaction partners and of the substance to be determined as well as of the enzyme. Further, ingredients such as proteins and detergents~to eliminate unspecific reactions, to reduce inhibiting influences or to enhance activation can be added to the incubation solution.
The novel method in accordance wlth the present invention is extraordinarily sensitive and is distinguished by its simplicity in operation.
The following Examples illustrate the invention.
Example 1 -Quantitative determination of CEA in plasma of patients with a monoclonal CEA antibody and a customary CEA antibody (goat) Into the requisite number of test tubes (10 x 75 mm) are in each case pipetted 0.2 ml of test solution (0.2 mol/l of N2H2P04/Na2HP04, pH 6.5 with 2 g/l of bovine serum albumin, 20~ of normal goat serum and 0.2 ~g/ml of goat--anti-CEA-peroxidase conjugate), 0.050 ml of the patient's plasma to be analysed or of the CEA standard (0 ng/ml of CEA, 2.5 ng/ml of CEA, 10 ng/ml of CEA and 20 ng/ml of CEA) and of the CEA control serum (5.0 ng/ml of CEA + 1.0 ng/ml) are admixed, in each case there is added a polystyrene sphere (0 = 6.5 mm) sensitised with monoclonal mouse anti-CEA~ and the mixture is incubated at 37C for 16 hours. Subsequently, the polystyrene spheres are washed three times with in each case 2-5 ml of distilled water, transferred into in each case 0.5 ml of substrate buffer for the determination of the activity of the peroxidase (0.1 mol/l of potassium citrate buffer of pH 5.0 with 6 mmol/1 of hydrogen peroxide and 20 mmol/l of o-phenylenediamine) and incubated for 30 minutes at room temperature (22C). In order to stop the peroxidic activity and to intensify the colour intensity 2.0 ml of lN
l~()S~
- 9 - ~
hydrochloric acid are admixed and withi~ 30 minutes the extinction is measured photometrically at the wa-~elength 492 nm. The values of a CEA determination are compiled in Table I and compared with the values which have been obtained with the radioimmunoassay of ROCHE.
*The preparation of the monoclonal mouse anti-CEA
is carried out in analogy to the method described in Journal of Immunological Methods, 32 (1980) 297-304, there being used as the starting cell line for the fusion the myeloma line Sp 2/01-AG which is deposited at ATCC under No. CRL 8006. The fusion is carried out with spleen cells of mice which ha~e been immunised with CEA. The immunisation of the mice was carried out in analog~ to Table 1 of the aforementioned publication, the first two immunisations being carried out with in each case 50 ~g of CEA, immunisations 3 and 4 being omitted, immunisation 5 being carried out with 50 ~ of CEA and immunisations 6-8 being carried out with in - each case 200 ~g of CEA.
~1~S~6 Table I
Sample material ~E492 nm/RT/30 min-CEA s tandard 0 ng/ml CEA O. 103
It has hitherto been the opinion that, in the case of enzyme-immune processes, the immunological reaction of the substance to be determined had to be effected with the two corresponding immunologically active reaction partners successively in a first and in a second reaction step.
In the scope of the present invention it has now surprisingly been found that, contrary to the hitherto existing opinion, it is possible to work in e~fect in a "one-pot process", with the substance to be determined being reacted simultaneously with both immunologically active reaction partners during a single incubation.
The present invention is accordingly concerned with an enzyme-immune process for the detection and determination of a substance with an immunologically active reaction partner, which is marked with an enzyme, as well as an immunologically active reaction partner, which is or will be bound to a water-insoluble carrier, by incubation of the substance to be determined with the immunologically active reaction partners, subsequent separation of solid and liquid phases and measurement of the extent of the enzyme marking either in the solid or in the liquid phase as the extent of the amount of the substance to be determined, which is characterised in that for the immunological reaction the substance to be determined as well as the immunologically active reaction partners are incubated from the outset and together and only once.
The substance to be determined can be any constituents in physiological fluids, cell extracts or tissue extracts for which there are present or can be formed immunological reaction partners, and which possess two or more immuno-logically active sites. Such substances include peptides, ~l~O';~
proteins, lipoproteins, glycoproteins, sterols, steroids, lipoids, nucleic acids, enzymes, ho:rmones, polysaccharides and alkaloids, Preferred immunologically active substances are natural antigens such as hormones, enzymes, organ--specific antigens, connective tissue components, blood cell antigens, plasma proteins and pathological globulins.
Especially preferred substances are carcinoembryonic antigen (CEA) as well as human choriongonadotropin (HCG).
Thus the present invention provides an immunoassay process for determining substances having at least two immuno-logically active sites comprising:
a) preparing a mixture of (i) a substance having at least two immunologically active sites or epitopes, (ii) an immunologically active reaction partner having an enzyme marker, and (iii) an immunologically active reaction partner which is or will be bound to a water insoluble carrier, b) incubating said mixture, c) separating the solid and liquid phase, d) determining the amount of substance by measuring the extent of the enzyme marking in the solid or liquid phase, the improvement consisting in e) using different monoclonal antibodies as the immunologi-cally active reaction partners, orf) using a monoclonal antibody and an antibody from a dif-ferent animal species as the immunologically active ~-~ reaction partners.
116~S~j - 3a -In the method in accordance with the invention the immunologically active reaction partner, which is marked with an enzyme, serves as the indicator of the immunological reac-tion. Preferred enzymes are alkaline phosphatase (EC 3.1.3.1), malate dehydrogenase (EC 1.11.37), glucose-6-phosphate de-hydrogenase (EC 1.1.1.49), glucose oxidase (EC 1.1.3.4), gluco-amylase (EC 3.2.1.3), galactosldase (EC 3.2.1.22/3.2.1.23) and acetylcholine esterase (EC 3.1.1.7). Peroxidase from horse-radish (EC 1.11.1.7) is an especially preferred enzyme label.
The enzyme as the indicator of the immunological reaction is measured in the liquid phase or, preferably, in the solid phase according to known methods and is a measurement for the amount of the substance to be determined. When the label is peroxidase from horseradish the amount of enzyme is preferably measured on the basis of the catalytic activity present, which is determined with the aid of hydrogen peroxide and o-phenylenediamine as the redox indicator. In ., ,6 this case, after a catalytic reaction lasting for 30 minutes the colour intensity of the oxid:Lsed redox indicator is measured photometrically.
The second immunologically active reaction partner serves for ~he separation of the substance to be determined from the analytical sample. For this separation step the second immunologically active reaction partner can be bound to a water-insoluble carrier from the outset or can be bound to such a carrier during or after the immunological reaction.
Suitable water-insoluble carriers for the second immunologicaliy active reaction partner are organic and inorganic polymers [amylases, dextrans, natural or modified celluloses, polyacrylamides, agaroses, magnetite, porous glass powder, polyvinylidene fluoride (Kynar) and latex], the inner wall of test vessels (test tubes, titre plates or cuvettes of glass or artificial materials) as well as the surface of solid bodies (rods of glass and artificial material, rods with terminal thickening, rods with terminal lobes or lamellae). Spheres of glass and artificial material are especially suitable carriers for the method in accordance with the invention.
~ he second immunologically active reaction partner can be bound to the water-insoluble carrier physically (a~sorptively) or chemically or with the aid of a further reaction partner, which, in turn, is bound to a carrier.
It is essential for the method in accordance with the invention that the substance to be determined possesses at least two immunologically active sites (epitopes), which can be recognised by and react with the two immunologically active reaction partners, the reaction partner which is bound to a carrier and the reaction partner which is marked with an enzyme. As the immunologically active reaction partners there are preferably used two different components, which indeed both react with the substance to be determined, but each cf which is directed to different immunologically active sites. For the determinatlon of antigens there are especially suitable two antibodies for the particular antigen, the antibodies having been produced in two different animal species and being directed towards different epitopes of the antigen. The combination of antibodies of different - clones or of monoclonal antibodies and antibodies from a different animal species is particularly suitable.
For the immunological reaction the sample with the substance to be determined can be used directly or can be pre-diluted or pre-treated in a suitable manner. For the pre-treatment the substance to be determined can be isolated, enriched or freed from troublesome ingredients.
According to the process in accordance with the invention, the substance to be determined is rea~ted simultaneously with the enzyme-marked as well as the carrier--bound immunologically active reaction partner. The sequence in which the reagents are added is governed by the type of carrier system chosen. When a sensitised plastic sphere is used, the enzyme-marked reaction partner and the substance to be determined are initially placed together in a suitable test tube and subsequently the sphere sensitised with the other reaction partner is added.
The immunological reaction is carried out in a suitable buffer system so as to maintain an optimum pH-value, which can lie between 4 and 9. Preferred buffers are, for example, acetate buffer, citrate buffer, phosphate buffer, tris buffer, triethanolamine buffer, borate buffer or glycine buffer. Buffer mixtures can also be used.
The immunological reaction is preferably carried out at a temperature between 0C and 55C. Normally, the immunological reaction velocity increases with higher temperatures, whereby under otherwise similar test conditions the equilibrium is achieved more rapidly.
The incubation of the substance to be determined with the enzyme-marked reaction partner and the carrier-bound reaction partner can be carried out until the equilibrium - 25 has been reached. The immunological reaction can, however, 'iÇ;6 also be stopped at an earlier point in time by separating the solid and the liquid phase after a defined incubation period and measuring the extent of the enzyme marking either in the liquid phase or in the solid phase.
In the immunological reaction precautions can be taken to stabilize the immunological activity of the reaction partners and of the substance to be determined as well as of the enzyme. Further, ingredients such as proteins and detergents~to eliminate unspecific reactions, to reduce inhibiting influences or to enhance activation can be added to the incubation solution.
The novel method in accordance wlth the present invention is extraordinarily sensitive and is distinguished by its simplicity in operation.
The following Examples illustrate the invention.
Example 1 -Quantitative determination of CEA in plasma of patients with a monoclonal CEA antibody and a customary CEA antibody (goat) Into the requisite number of test tubes (10 x 75 mm) are in each case pipetted 0.2 ml of test solution (0.2 mol/l of N2H2P04/Na2HP04, pH 6.5 with 2 g/l of bovine serum albumin, 20~ of normal goat serum and 0.2 ~g/ml of goat--anti-CEA-peroxidase conjugate), 0.050 ml of the patient's plasma to be analysed or of the CEA standard (0 ng/ml of CEA, 2.5 ng/ml of CEA, 10 ng/ml of CEA and 20 ng/ml of CEA) and of the CEA control serum (5.0 ng/ml of CEA + 1.0 ng/ml) are admixed, in each case there is added a polystyrene sphere (0 = 6.5 mm) sensitised with monoclonal mouse anti-CEA~ and the mixture is incubated at 37C for 16 hours. Subsequently, the polystyrene spheres are washed three times with in each case 2-5 ml of distilled water, transferred into in each case 0.5 ml of substrate buffer for the determination of the activity of the peroxidase (0.1 mol/l of potassium citrate buffer of pH 5.0 with 6 mmol/1 of hydrogen peroxide and 20 mmol/l of o-phenylenediamine) and incubated for 30 minutes at room temperature (22C). In order to stop the peroxidic activity and to intensify the colour intensity 2.0 ml of lN
l~()S~
- 9 - ~
hydrochloric acid are admixed and withi~ 30 minutes the extinction is measured photometrically at the wa-~elength 492 nm. The values of a CEA determination are compiled in Table I and compared with the values which have been obtained with the radioimmunoassay of ROCHE.
*The preparation of the monoclonal mouse anti-CEA
is carried out in analogy to the method described in Journal of Immunological Methods, 32 (1980) 297-304, there being used as the starting cell line for the fusion the myeloma line Sp 2/01-AG which is deposited at ATCC under No. CRL 8006. The fusion is carried out with spleen cells of mice which ha~e been immunised with CEA. The immunisation of the mice was carried out in analog~ to Table 1 of the aforementioned publication, the first two immunisations being carried out with in each case 50 ~g of CEA, immunisations 3 and 4 being omitted, immunisation 5 being carried out with 50 ~ of CEA and immunisations 6-8 being carried out with in - each case 200 ~g of CEA.
~1~S~6 Table I
Sample material ~E492 nm/RT/30 min-CEA s tandard 0 ng/ml CEA O. 103
2.5 ng/ml CEA 0.330 10.0 ng/ml CEA O . 9 78 20~0 ng/ml CEA 1.850 I
CEA control serum 5.0 ng/ml CEA O . 540 Process in accordance Patient's plasm ROCHE RIA test with the invention No. 72120.6 ng/ml 1.2 ng/ml No. 71882.2 ng/ml 1.0 ng/ml No. 72201.2 ng/ml 1.4 ng/ml No. 72181.2 ng/ml 1.3 ng/ml No. 72342.5 ng/ml 2.7 ng/ml No. 72492.4 ng/ml 2.0 ng/ml No. 72032.3 ng/ml 2.0 ng/ml No. 72233.0 ng/ml 3.3 ng/ml No. 72473.1 ng/ml 2.8 ng/ml No. 72584.6 ng/ml 4.3 ng/ml No. 72154.9 ng/ml 5.5 ng/ml No. 72195.0 ng/ml 5.9 ng/mg No. 81808.6 ng/ml 8.5 ng/ml No. 724811.2 ng/ml 10.7 ng/ml No. 726214.2 ng/ml 14.3 ng/ml No. 720115.6 ng/ml 15.3 ng/ml ~1605~;6 Values below 2.5 ng/ml of CEA lie in the normal range, while values above 2.5 ng/ml lie in the patholo~ical range.
Example 2 Quantitative determination of CEA in plasma of patients with CE~ antibodies from two different animal s ecies P
(goat and guinea pig) Into the requisite number of test tubes (lO x 75 mm) are in each case pipetted 0.2 ml of test solution (0.2 mol/l of NaH2P04/Na2HP04, pH 6.5 with 2 g/l of bovine serum albumin, 20~ of normal goat serum and 0.2 ~g/ml of goat--anti-CEA-peroxidase conjugate), 0.050 ml of the patient's plasma to be analysed or of the CEA standard (0 ng/ml of CEA, 2.5 ng/ml of CEA, 10 ng/ml of CEA and 20 ng/ml of CEA) and of the CEA control serum (5.0 ng/ml of CEA + l.0 mg/ml) are admixed, in each case there is added a polystyrene sphere (0 = 6.5 mm) sensitised with guinea pig-anti-CEA and the mixture is incubated at 37C for 16 hours. Subsequently, the polystyrene spheres are washed three times with in each case 2-5 ml of distilled water, transferred into in each case 0.5 ml of substrate buffer for the determination of the activity of the peroxidase (0.1 mol/l of potassium citrate buffer of pH 5.0 with 6 mmol/l of hydrogen peroxide and 20 mmoljl of o-phenylenediamine) and incubated for 30 minutes US~
at room temperature (22C~. In order to stop the peroxidic activity and to intensify the colour intensity 2.0 ml of lN hydrochloric acid are admixed and within 30 minutes the extinction is measured photometrically at the wavelength 492 nm. The values of a CEA determination are compiled in Table II and compared with the values which have been obtained with the radioimmunoassay of ROCHE.
;6 Table II
Sample material _ ~E492 nm/RT/30 min.
CEA standard O ng/ml CEA 0.058 2.5 ng/ml CEA 0.270 10.0 ng/ml CEA 0.830 20.0 ng/ml CEA 1.515 CEA control serum 5.0 ng/ml CEA 0.470 Process in accordance Patient's plasma ROCHE RIA test with the invention No. 72201.2 ng/ml 1.3 ng/ml No. 72181.2 ng/ml 1.3 ng/ml No. 72342.5 ng/ml 2.4 ng/ml No. 72492.4 ng/ml 2.2 ng/ml No. 72032.3 ng/ml 2.2 ng/ml No. 72233.0 ng/ml 3.0 ng/ml No. 72473.1 ng/ml 3.2 ng/ml No. 72584.6 ng/ml 4.5 ng/ml No. 72154.9 ng/ml 5.3 ng/ml No. 72195.0 ng/ml 5.6 ng/ml No. 81808.6 ng/ml 8.5 ng/ml No. 724811.2 ng/ml10.9 ng/ml No. 726214.2 ng/ml14.2 ng/ml No. 720115.6 ng/ml15.0 ng/ml Values below 2.5 ng/ml of OE A lie in the normal range, while values above 2.5 ng/ml lie in the pathological range.
Example 3 Quantitative determination of CEA in plasma of Patients with monoclonal CEA antibodies from two different clones _ Into the requisite number of test tubes (10 x 75 mm) are in each case pipetted 0.2 ml of test solution (0.2 mol/l of NaH2P04/Na2HP04 pH 6.5 with 2 g/l of bovine lo serum albumin, 20% of normal goat serum and 0.15 ~g/ml of monoclonal mouse-anti-CEA peroxidase conjugate*), 0.050 ml of the patient's plasma to be analysed or of the CEA
standard (0 ng/ml of CEA, 2.5 ng/ml of CEA, 10 ng/ml of CEA and 20 ng/ml of CEA) and of the CEA control serum (5.0 ng/ml of CEA + 1.0 ng/ml) are admixed, in each case there is added a polystyrene sphere (0 = 6.5 mm) sensitised with monoclonal mouse anti-CEA* and the mixture is incubated at 37C for 16 hours. Subsequently, the polystyrene spheres are washed three times with in each case 2-5 ml of distilled water, transferred into in each case 0.5 ml of substrate buffer for the determination of the activity of the peroxidase (0.1 mol/l of potassium citrate buffer of pH -5.0 with 6 mmol/l of hydrogen peroxide and 20 mmol/l ~6V~i6 of o-phenylenediamine) and incubated for 30 minutes at room temperature ~22C). In order to stop the peroxidic activity and to intensify the colour intensity 2.0 ml of lN
hydrochloric acid are admixed and within 30 minutes the extinction is measured photometrically at the wavelength 492 nm. The values of a CEA determination are compiled in Table III and compared with the values which have been obtained with the radioimmunoassay of ROCHE.
*The preparation of the monoclonal mouse anti-CEA is carried out in analogy to the method described in Journal of Immunological Methods, 32 (1980) 297-304 ! there being used as the starting cell line for the fusion the myeloma line Sp 2/01-Ag which is deposited at ATCC under No CRL 8006.
The fusion is carried 01~t with spleen cells of mice which have been immunised with CEA. The immunisation of the mice was carried out in analogy to Table 1 of the aforementioned publication, the first two immunisations being carried out with in each case 50 ~g of CEA, immunisations 3 and 4 being omitted, immunisation S being carried out with 50 ~g of CEA
and immunisations 6-8 being carried out with in each case 200 ~g of CEA. There are used two different, suitable monoclonal antibodies which are directed towards different epitopes of the OE A antigen.
i~6~S~6 Table ~II
Sample material ~E492 nmJRT/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 _ _ .
CEA control serum 5.0 ng/ml CEA 0.510 _ atient's plasma ROCHE RIA test Process in accord-ance with the invention No. 72201.2 ng/ml Ø8 ng/ml No. 72342.5 ng/ml 3.0 ng/ml No. 72233.0 ng/ml 3.5 ng/ml No. 72473.1 ng/ml 3.2 ng/ml No. 72584.6 ng/ml 4.4 ng/ml No. 72154.9 ng/ml 5.0 ng/ml No. 72195.0 ng/ml 5.4 ng/ml No. 81808.6 ng/ml 8.6 ng/ml No. 724811.2 ng/ml 11.0 ng/ml .
No. 726214.2 ng/ml 14.0 ng/ml No. 720115.6 ng/ml 16.0 ng/ml _ _ S~SF~
Values below 2.5 ng/ml of CEA lie in the normal range, while values above 2.5 ng/ml lie in the pathological range.
Example 4 Quantitative determination of HCG in serum/plasma/urine:
Into the requisite number of test tubes (10 x 75 mm) are in each case pipetted 0.2 ml of test solution (0.1 mol/l of NaH2P04lNa2HP04, pH 7.0 with 2 g/l of bovine serum albumin, and 1.0 ~g/ml of monoclonal mouse-anti-HCG--peroxidase conjugate*), 0~050 ml Of the patient's plasma to be analysed or of the HCG standard (0, 25, 50, 100 and 250 mIU/ml of HCG) is admixed, in each case there is added a polystyrene sphere (0 = 6.5 mm) sensitised with rabbit--anti-HCG and the mixture is incuba~ed at room temperature for 16 hours in a water-saturated at~osphere. Subsequently, the polystyrene spheres are washed three times with in each case 2-5 ml of distilled water, transferred into in each case 0.5 ml of substrate buffer for the determination of the activity of the peroxidase (0.1 mol/l of potassium citrate buffer of pH 5.0 with 6 mmol/l of hydrogen peroxide and 20 mmol/l of o-phenylenediamine) and incubated for 30 minutes at room temperature tl6-30C). In order to ;S6 stop the peroxidic activity and to intensify the colour intensity 2.0 ml of lN hydrochloric acid are admixed and within 30 minutes the extinction is measured photometrically at the wavelength 492 nm. The values of a HCG determination in serum and urine are compiled int~e following Tabie.
*The preparation of the monoclonal anti-HCG can be carried out according to one of the methods described in Journal of Immunological Methods, 32 (1980) 297-304.
HCG determination in serum and in urine 1. Standard curves ~E /30 min./RT
492 nm mIU HCG/ml Serum Urine 0 0.185 0.385 0.385 0.525 0.530 0.720 15100 0.830 1.05 250 1.185 1 1.59 - lg -2. _atient1s samples Sample No. ~E492nm/3 min./RT = mIU HCG/ml sample .
Urine N-1032 E 0.375 0 " 58-418 0.575 (x 50) * 1500 " 58414 0.775 (x 100) * 5500 " 58416 0.810 (x 500) * 32000 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 * Dilution of the sample onversion: 1 ng of pure HCG corresponds to ca. 10 mIU
of HCG.
Example 5 Quantitative determination of HCG in serum with monoclonal HCG antibodies from two different clones Into the requisite number of test tubes (10 x 75 mm) are in each case pipetted 0.2 ml of test solution (0.1 mol/l of NaH2P04/Na2HP04,pH 7.0 with 2 g/l of bovine serum albumin and 1.0 ~Ig/ml of monoclonal mouse--anti-HCG-peroxidase conjugate*), 0.050 ml of the patient's plasma to be analysed or of the HCG standard (0, 25, 50, 100 and 200 mIU/ml of HCG) are admixed, in each case there is added a polystyrene sphere (0 = 6.5 mm) sensitised with monoclonal mouse-anti-~CG* and the mixture is incubated, for example, at 37C for. 2 hours. Subsequently, the polystyrene spheres are washed three times with in each case 2-5 ml of distilled water, transferred into in each case 0.5 ml of substrate buffer for the determination of the activity of the peroxidase (0.1 mol/l of potassium citrate buffer of pH 5.0 with 6 mmol/l of hydrogen peroxide and 20 mmol/l of o-phenylenediamine) and incubated for 30 minutes at room temperature (16-30C). In oraer to stop the peroxidic activity and to intensify the colour intensity 1.0 ml of 2N hydrochloric acid are admixed and ~ithin 30 minutes the extinction is measured photometrically at the wavelength 492 nm. The values of HCG determinations in serum are compiled in the following Table.
*The preparatisn of the monoclonal mouse HCG antibody is carried out according to the method described in Journal of Immunological Methods, 32 (1980) 297-304. There are used two different, suitable monoclonal antibodies which are directed towards different epitopes of the HCG antigen.
HCG determination in human serum (average values from duplicate determinations) 1. Standard curves GE 492 nm/30 min./RT
10 mIU/ml HCG 1) Serum 2) Plasma 3) Urine 4) Buffer I
0 0.038 0.054 0.158 0.180 0.226 _ _ _ 0.475 o,544 0,557 0.716 100 0.905 0.955 0.970 1.40 200 1.75 1.55 1.90 2044 l~t`~
The HCG values of the examples of patient's sera compiled below were read-off from standard curve 1). Standard curves 2), 3) and 4) were established on another day with new HCG
standards.
2. Patient's sera ~E 492 nm/30 min./RT mIU/ml HCG
Serum measured from curve 1) . ~
Pool 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 o.975 (1:50)5,450 No. 3240 1.06 (1:20) 2,280 No. 4418 1.475 (1:1000)167,000
CEA control serum 5.0 ng/ml CEA O . 540 Process in accordance Patient's plasm ROCHE RIA test with the invention No. 72120.6 ng/ml 1.2 ng/ml No. 71882.2 ng/ml 1.0 ng/ml No. 72201.2 ng/ml 1.4 ng/ml No. 72181.2 ng/ml 1.3 ng/ml No. 72342.5 ng/ml 2.7 ng/ml No. 72492.4 ng/ml 2.0 ng/ml No. 72032.3 ng/ml 2.0 ng/ml No. 72233.0 ng/ml 3.3 ng/ml No. 72473.1 ng/ml 2.8 ng/ml No. 72584.6 ng/ml 4.3 ng/ml No. 72154.9 ng/ml 5.5 ng/ml No. 72195.0 ng/ml 5.9 ng/mg No. 81808.6 ng/ml 8.5 ng/ml No. 724811.2 ng/ml 10.7 ng/ml No. 726214.2 ng/ml 14.3 ng/ml No. 720115.6 ng/ml 15.3 ng/ml ~1605~;6 Values below 2.5 ng/ml of CEA lie in the normal range, while values above 2.5 ng/ml lie in the patholo~ical range.
Example 2 Quantitative determination of CEA in plasma of patients with CE~ antibodies from two different animal s ecies P
(goat and guinea pig) Into the requisite number of test tubes (lO x 75 mm) are in each case pipetted 0.2 ml of test solution (0.2 mol/l of NaH2P04/Na2HP04, pH 6.5 with 2 g/l of bovine serum albumin, 20~ of normal goat serum and 0.2 ~g/ml of goat--anti-CEA-peroxidase conjugate), 0.050 ml of the patient's plasma to be analysed or of the CEA standard (0 ng/ml of CEA, 2.5 ng/ml of CEA, 10 ng/ml of CEA and 20 ng/ml of CEA) and of the CEA control serum (5.0 ng/ml of CEA + l.0 mg/ml) are admixed, in each case there is added a polystyrene sphere (0 = 6.5 mm) sensitised with guinea pig-anti-CEA and the mixture is incubated at 37C for 16 hours. Subsequently, the polystyrene spheres are washed three times with in each case 2-5 ml of distilled water, transferred into in each case 0.5 ml of substrate buffer for the determination of the activity of the peroxidase (0.1 mol/l of potassium citrate buffer of pH 5.0 with 6 mmol/l of hydrogen peroxide and 20 mmoljl of o-phenylenediamine) and incubated for 30 minutes US~
at room temperature (22C~. In order to stop the peroxidic activity and to intensify the colour intensity 2.0 ml of lN hydrochloric acid are admixed and within 30 minutes the extinction is measured photometrically at the wavelength 492 nm. The values of a CEA determination are compiled in Table II and compared with the values which have been obtained with the radioimmunoassay of ROCHE.
;6 Table II
Sample material _ ~E492 nm/RT/30 min.
CEA standard O ng/ml CEA 0.058 2.5 ng/ml CEA 0.270 10.0 ng/ml CEA 0.830 20.0 ng/ml CEA 1.515 CEA control serum 5.0 ng/ml CEA 0.470 Process in accordance Patient's plasma ROCHE RIA test with the invention No. 72201.2 ng/ml 1.3 ng/ml No. 72181.2 ng/ml 1.3 ng/ml No. 72342.5 ng/ml 2.4 ng/ml No. 72492.4 ng/ml 2.2 ng/ml No. 72032.3 ng/ml 2.2 ng/ml No. 72233.0 ng/ml 3.0 ng/ml No. 72473.1 ng/ml 3.2 ng/ml No. 72584.6 ng/ml 4.5 ng/ml No. 72154.9 ng/ml 5.3 ng/ml No. 72195.0 ng/ml 5.6 ng/ml No. 81808.6 ng/ml 8.5 ng/ml No. 724811.2 ng/ml10.9 ng/ml No. 726214.2 ng/ml14.2 ng/ml No. 720115.6 ng/ml15.0 ng/ml Values below 2.5 ng/ml of OE A lie in the normal range, while values above 2.5 ng/ml lie in the pathological range.
Example 3 Quantitative determination of CEA in plasma of Patients with monoclonal CEA antibodies from two different clones _ Into the requisite number of test tubes (10 x 75 mm) are in each case pipetted 0.2 ml of test solution (0.2 mol/l of NaH2P04/Na2HP04 pH 6.5 with 2 g/l of bovine lo serum albumin, 20% of normal goat serum and 0.15 ~g/ml of monoclonal mouse-anti-CEA peroxidase conjugate*), 0.050 ml of the patient's plasma to be analysed or of the CEA
standard (0 ng/ml of CEA, 2.5 ng/ml of CEA, 10 ng/ml of CEA and 20 ng/ml of CEA) and of the CEA control serum (5.0 ng/ml of CEA + 1.0 ng/ml) are admixed, in each case there is added a polystyrene sphere (0 = 6.5 mm) sensitised with monoclonal mouse anti-CEA* and the mixture is incubated at 37C for 16 hours. Subsequently, the polystyrene spheres are washed three times with in each case 2-5 ml of distilled water, transferred into in each case 0.5 ml of substrate buffer for the determination of the activity of the peroxidase (0.1 mol/l of potassium citrate buffer of pH -5.0 with 6 mmol/l of hydrogen peroxide and 20 mmol/l ~6V~i6 of o-phenylenediamine) and incubated for 30 minutes at room temperature ~22C). In order to stop the peroxidic activity and to intensify the colour intensity 2.0 ml of lN
hydrochloric acid are admixed and within 30 minutes the extinction is measured photometrically at the wavelength 492 nm. The values of a CEA determination are compiled in Table III and compared with the values which have been obtained with the radioimmunoassay of ROCHE.
*The preparation of the monoclonal mouse anti-CEA is carried out in analogy to the method described in Journal of Immunological Methods, 32 (1980) 297-304 ! there being used as the starting cell line for the fusion the myeloma line Sp 2/01-Ag which is deposited at ATCC under No CRL 8006.
The fusion is carried 01~t with spleen cells of mice which have been immunised with CEA. The immunisation of the mice was carried out in analogy to Table 1 of the aforementioned publication, the first two immunisations being carried out with in each case 50 ~g of CEA, immunisations 3 and 4 being omitted, immunisation S being carried out with 50 ~g of CEA
and immunisations 6-8 being carried out with in each case 200 ~g of CEA. There are used two different, suitable monoclonal antibodies which are directed towards different epitopes of the OE A antigen.
i~6~S~6 Table ~II
Sample material ~E492 nmJRT/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 _ _ .
CEA control serum 5.0 ng/ml CEA 0.510 _ atient's plasma ROCHE RIA test Process in accord-ance with the invention No. 72201.2 ng/ml Ø8 ng/ml No. 72342.5 ng/ml 3.0 ng/ml No. 72233.0 ng/ml 3.5 ng/ml No. 72473.1 ng/ml 3.2 ng/ml No. 72584.6 ng/ml 4.4 ng/ml No. 72154.9 ng/ml 5.0 ng/ml No. 72195.0 ng/ml 5.4 ng/ml No. 81808.6 ng/ml 8.6 ng/ml No. 724811.2 ng/ml 11.0 ng/ml .
No. 726214.2 ng/ml 14.0 ng/ml No. 720115.6 ng/ml 16.0 ng/ml _ _ S~SF~
Values below 2.5 ng/ml of CEA lie in the normal range, while values above 2.5 ng/ml lie in the pathological range.
Example 4 Quantitative determination of HCG in serum/plasma/urine:
Into the requisite number of test tubes (10 x 75 mm) are in each case pipetted 0.2 ml of test solution (0.1 mol/l of NaH2P04lNa2HP04, pH 7.0 with 2 g/l of bovine serum albumin, and 1.0 ~g/ml of monoclonal mouse-anti-HCG--peroxidase conjugate*), 0~050 ml Of the patient's plasma to be analysed or of the HCG standard (0, 25, 50, 100 and 250 mIU/ml of HCG) is admixed, in each case there is added a polystyrene sphere (0 = 6.5 mm) sensitised with rabbit--anti-HCG and the mixture is incuba~ed at room temperature for 16 hours in a water-saturated at~osphere. Subsequently, the polystyrene spheres are washed three times with in each case 2-5 ml of distilled water, transferred into in each case 0.5 ml of substrate buffer for the determination of the activity of the peroxidase (0.1 mol/l of potassium citrate buffer of pH 5.0 with 6 mmol/l of hydrogen peroxide and 20 mmol/l of o-phenylenediamine) and incubated for 30 minutes at room temperature tl6-30C). In order to ;S6 stop the peroxidic activity and to intensify the colour intensity 2.0 ml of lN hydrochloric acid are admixed and within 30 minutes the extinction is measured photometrically at the wavelength 492 nm. The values of a HCG determination in serum and urine are compiled int~e following Tabie.
*The preparation of the monoclonal anti-HCG can be carried out according to one of the methods described in Journal of Immunological Methods, 32 (1980) 297-304.
HCG determination in serum and in urine 1. Standard curves ~E /30 min./RT
492 nm mIU HCG/ml Serum Urine 0 0.185 0.385 0.385 0.525 0.530 0.720 15100 0.830 1.05 250 1.185 1 1.59 - lg -2. _atient1s samples Sample No. ~E492nm/3 min./RT = mIU HCG/ml sample .
Urine N-1032 E 0.375 0 " 58-418 0.575 (x 50) * 1500 " 58414 0.775 (x 100) * 5500 " 58416 0.810 (x 500) * 32000 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 * Dilution of the sample onversion: 1 ng of pure HCG corresponds to ca. 10 mIU
of HCG.
Example 5 Quantitative determination of HCG in serum with monoclonal HCG antibodies from two different clones Into the requisite number of test tubes (10 x 75 mm) are in each case pipetted 0.2 ml of test solution (0.1 mol/l of NaH2P04/Na2HP04,pH 7.0 with 2 g/l of bovine serum albumin and 1.0 ~Ig/ml of monoclonal mouse--anti-HCG-peroxidase conjugate*), 0.050 ml of the patient's plasma to be analysed or of the HCG standard (0, 25, 50, 100 and 200 mIU/ml of HCG) are admixed, in each case there is added a polystyrene sphere (0 = 6.5 mm) sensitised with monoclonal mouse-anti-~CG* and the mixture is incubated, for example, at 37C for. 2 hours. Subsequently, the polystyrene spheres are washed three times with in each case 2-5 ml of distilled water, transferred into in each case 0.5 ml of substrate buffer for the determination of the activity of the peroxidase (0.1 mol/l of potassium citrate buffer of pH 5.0 with 6 mmol/l of hydrogen peroxide and 20 mmol/l of o-phenylenediamine) and incubated for 30 minutes at room temperature (16-30C). In oraer to stop the peroxidic activity and to intensify the colour intensity 1.0 ml of 2N hydrochloric acid are admixed and ~ithin 30 minutes the extinction is measured photometrically at the wavelength 492 nm. The values of HCG determinations in serum are compiled in the following Table.
*The preparatisn of the monoclonal mouse HCG antibody is carried out according to the method described in Journal of Immunological Methods, 32 (1980) 297-304. There are used two different, suitable monoclonal antibodies which are directed towards different epitopes of the HCG antigen.
HCG determination in human serum (average values from duplicate determinations) 1. Standard curves GE 492 nm/30 min./RT
10 mIU/ml HCG 1) Serum 2) Plasma 3) Urine 4) Buffer I
0 0.038 0.054 0.158 0.180 0.226 _ _ _ 0.475 o,544 0,557 0.716 100 0.905 0.955 0.970 1.40 200 1.75 1.55 1.90 2044 l~t`~
The HCG values of the examples of patient's sera compiled below were read-off from standard curve 1). Standard curves 2), 3) and 4) were established on another day with new HCG
standards.
2. Patient's sera ~E 492 nm/30 min./RT mIU/ml HCG
Serum measured from curve 1) . ~
Pool 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 o.975 (1:50)5,450 No. 3240 1.06 (1:20) 2,280 No. 4418 1.475 (1:1000)167,000
Claims (20)
1. An immunoassay process for determining substances having at least two immunologically active sites comprising:
a) preparing a mixture of (i) a substance having at least two immunologically active sites or epitopes, (ii) an immunologically active reaction partner having an enzyme marker, and (iii) an immunologically active reaction partner which is or will be bound to a water insoluble carrier, b) incubating said mixture, c) separating the solid and liquid phase, d) determining the amount of substance by measuring the extent of the enzyme marking in the solid or liquid phase, the improvement consisting in e) using different monoclonal antibodies as the immunologically active reaction partners, or f) using a monoclonal antibody and an antibody from a different animal species as the immunologically active reaction partners.
a) preparing a mixture of (i) a substance having at least two immunologically active sites or epitopes, (ii) an immunologically active reaction partner having an enzyme marker, and (iii) an immunologically active reaction partner which is or will be bound to a water insoluble carrier, b) incubating said mixture, c) separating the solid and liquid phase, d) determining the amount of substance by measuring the extent of the enzyme marking in the solid or liquid phase, the improvement consisting in e) using different monoclonal antibodies as the immunologically active reaction partners, or f) using a monoclonal antibody and an antibody from a different animal species as the immunologically active reaction partners.
2. A process according to claim 1, characterised in that the substance to be determined is an antigen.
3. A process according to claim 1, characterised in that the substance to be determined is CEA.
4. A process according to claim 3, characterised in that the immunologically active reaction partner, which is bound to a water-insoluble carrier, is monoclonal mouse-anti-CEA.
5. A process according to claim 4, characterised in that the immunologically active reaction partner, which is provided with a marking, is another monoclonal mouse-anti-CEA marked with an enzyme.
6. A process according to claim 4, characterised in that the immunologically active reaction partner, which is provided with a marking, is an enzyme-marked goat-anti-CEA.
7. A process according to claim 5 or 6, characterised in that the enzyme is peroxidase.
8. A process according to claim 1, characterised in that the antigen is HCG.
9. A process according to claim 8, characterised in that the immunologically active reaction partner, which is bound to a water-insoluble carrier, is rabbit-anti-HCG.
10. A process according to claim 9, characterised in that the immunologically active reaction partner, which is provided with the marking, is an enzyme-marked monoclonal mouse-anti-HCG.
11. A process as claimed in claim 10, characterised in that the enzyme is peroxidase.
12. A process according to claim 2, characterised in that the substance to be determined is CEA.
13. A process according to claim 12, characterised in that the immunologically active reaction partner, which is bound to a water-insoluble carrier, is monoclonal mouse-anti-CEA.
14. A process according to claim 13, characterised in that the immunologically active reaction partner, which is provided with a marking, is another monoclonal mouse-anti-CEA marked with an enzyme.
15. A process according to claim 13, characterised in that the immunologically active reaction partner, which is provided with a marking, is an enzyme-marked goat-anti-CEA.
16. A process according to claim 14 or 15, characterised in that the enzyme is peroxidase.
17. A process according to claim 2, characterised in that the antigen is HCG.
18. A process according to claim 17, characterised in that the immunologically active reaction partner, which is bound to a water-insoluble carrier, is rabbit-anti-HCG.
19. A process according to claim 18, characterised in that the immunologically active reaction partner, which is provided with the marking, is an enzyme-marked monoclonal mouse-anti-HCG.
20. A process as claimed in claim 19, characterised in that the enzyme is peroxidase.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CH3209/80 | 1980-04-25 | ||
| CH320980A CH642458A5 (en) | 1980-04-25 | 1980-04-25 | Immunological method |
| CH5898/80 | 1980-08-04 | ||
| CH589880A CH651396A5 (en) | 1980-08-04 | 1980-08-04 | Immunological method for detecting and determining carcinoembryonic antigen |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1160566A true CA1160566A (en) | 1984-01-17 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000373607A Expired CA1160566A (en) | 1980-04-25 | 1981-03-23 | Immunological determination method |
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|---|---|
| AT (1) | AT373398B (en) |
| AU (1) | AU542563B2 (en) |
| CA (1) | CA1160566A (en) |
| DE (1) | DE3115115A1 (en) |
| DK (1) | DK151399C (en) |
| FR (1) | FR2481318A1 (en) |
| GB (1) | GB2074727B (en) |
| IT (1) | IT1137344B (en) |
| NL (1) | NL187545B (en) |
| NO (1) | NO159620C (en) |
| SE (1) | SE460930B (en) |
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| US6406920B1 (en) | 1980-06-20 | 2002-06-18 | Inverness Medical Switzerland Gmbh | Processes and apparatus for carrying out specific binding assays |
| EP0111762B1 (en) * | 1980-06-20 | 1987-11-19 | Unilever Plc | Processes and apparatus for carrying out specific binding assays |
| JPS57501147A (en) * | 1980-07-16 | 1982-07-01 | ||
| 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 |
| US6881589B1 (en) | 1987-04-30 | 2005-04-19 | Bioveris Corporation | Electrochemiluminescent localizable complexes for assay compositions |
| US5935779A (en) * | 1988-11-03 | 1999-08-10 | Igen International Inc. | Methods for improved particle electrochemiluminescence assay |
| EP0328690B1 (en) * | 1987-08-25 | 1994-08-10 | Fuji Yakuhin Kogyo Kabushiki Kaisha | Method for diagnosing chronic articular rheumatism |
| DE3850962T2 (en) * | 1987-10-30 | 1994-12-22 | Fuji Yakuhin Kogyo Kk | DIAGNOSTIC PROCEDURE FOR LIVER CANCER. |
| 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 |
| US5705402A (en) * | 1988-11-03 | 1998-01-06 | Igen International, Inc. | Method and apparatus for magnetic microparticulate based luminescence assay including plurality of magnets |
| 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 |
| US5962218A (en) * | 1988-11-03 | 1999-10-05 | Igen International Inc. | Methods and apparatus for improved luminescence assays |
| US5779976A (en) * | 1988-11-03 | 1998-07-14 | Igen International, Inc. | Apparatus for improved luminescence assays |
| EP0485377B1 (en) * | 1988-12-12 | 1999-05-06 | Csl Limited | Solid phase immuno-assay with labelled conjugate |
| US5176999A (en) * | 1989-12-07 | 1993-01-05 | Eastman Kodak Company | Buffered wash composition, insolubilizing composition, test kits and method of use |
| ZA92803B (en) | 1991-02-06 | 1992-11-25 | Igen Inc | Method and apparatus for magnetic microparticulate based luminescene asay including plurality of magnets |
| 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. |
| JP3694894B2 (en) * | 1994-02-19 | 2005-09-14 | 生化学工業株式会社 | Measurement method and kit for normal aggrecan |
| EP1030179B1 (en) | 1998-09-01 | 2005-11-09 | Taiho Pharmaceutical Company Limited | Method for assaying human thymidylate synthase and assay kit |
| ATE365326T1 (en) | 1999-04-12 | 2007-07-15 | Sumitomo Chemical Co | METHOD FOR ANALYZING THE AMOUNT OF INTRA-ABDOMINAL FAT TISSUE |
| ES2675044T3 (en) | 2005-06-03 | 2018-07-06 | 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 |
| GB2107053A (en) * | 1980-06-20 | 1983-04-20 | Unilever Plc | Processes and apparatus for carrying out specific binding assays |
-
1981
- 1981-03-23 CA CA000373607A patent/CA1160566A/en not_active Expired
- 1981-04-06 DK DK155881A patent/DK151399C/en not_active IP Right Cessation
- 1981-04-13 IT IT21122/81A patent/IT1137344B/en active
- 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 NO NO811407A patent/NO159620C/en not_active IP Right Cessation
- 1981-04-24 GB GB8112730A patent/GB2074727B/en not_active Expired
- 1981-04-24 AT AT0186481A patent/AT373398B/en not_active IP Right Cessation
- 1981-04-24 AU AU69811/81A patent/AU542563B2/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| NL187545B (en) | 1991-06-03 |
| AU542563B2 (en) | 1985-02-28 |
| DK151399C (en) | 1988-09-05 |
| DK151399B (en) | 1987-11-30 |
| NO811407L (en) | 1981-10-26 |
| SE460930B (en) | 1989-12-04 |
| AT373398B (en) | 1984-01-10 |
| DK155881A (en) | 1981-10-26 |
| AU6981181A (en) | 1981-10-29 |
| NO159620B (en) | 1988-10-10 |
| IT1137344B (en) | 1986-09-10 |
| FR2481318B1 (en) | 1984-10-19 |
| SE8102558L (en) | 1981-10-26 |
| FR2481318A1 (en) | 1981-10-30 |
| NO159620C (en) | 1989-01-18 |
| GB2074727A (en) | 1981-11-04 |
| DE3115115C2 (en) | 1987-02-12 |
| DE3115115A1 (en) | 1982-02-04 |
| IT8121122A0 (en) | 1981-04-13 |
| GB2074727B (en) | 1983-11-30 |
| ATA186481A (en) | 1983-05-15 |
| NL8101860A (en) | 1981-11-16 |
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| MKEX | Expiry |