NL8302708A - IMMUNOLOGICAL METHOD AND REAGENT. - Google Patents

Description

V

r <t * VO 5003

Immunological measuring method and reagent.

The invention relates to a method for simultaneously measuring the total amount of two or more different substances present in one sample and a reagent therefor.

Recent advances in clinical research have enabled accurate diagnosis and treatment of various diseases by measuring a number of clinical points and properly assessing their results. With current immunochemical measurements, however, a single point test usually takes ± 1-4 days, while a large number of days is required to test a number of points, so that sometimes points needed are omitted for fear that a necessary medical treatment of the patient would be late. Under these circumstances, it is possible that the treatment of the patient is not always optimal.

In cancer, for example, it is believed that a measurement of various tumor markers are useful for early diagnosis, understanding of the extent of the disease and evaluation of the therapeutic effect, evaluation of a recurrence of the disease, etc. In such a case, it is more effectively and accurately to measure a number of tumor markers and to judge the results thereof than to judge based on a measurement of a single tumor marker. However, it takes many days to measure a number of tumor markers. Furthermore, the required amount of a sample (mainly serum, plasma, etc.) is thereby necessarily increased, which is unfavorable for the patient.

The invention now relates to a method for simultaneously measuring the total amount of two or more substances present in one sample by a single measuring treatment. The invention also relates to a reagent for simultaneously measuring the amount of two or more substances present in one sample by a single measuring treatment.

The invention thus relates to an immunological measuring method and a measuring reagent for simultaneously measuring the total amount of two or more substances to be measured, whereby a sample of substances to be measured is reacted with an insoluble antibody or anti-35 which does not contain two or more contains different antibodies or antigens, 8302708 t * -2- and determines the substances to be measured based on the observed immunological response.

Since according to the invention two or more substances to be measured can be measured simultaneously, the measuring time can be considerably reduced as well as the amount of sample required, compared to conventional methods, in which a number of substances to be measured were successively measured. The invention is illustrated by the drawing. Therein are: Fig. 1 to Fig. 4 graphs of the standard curves according to Example VI; 5 - 8 show graphs of the standard curves according to example X; Fig. 9 is a. graph about the standard curves in example XII; and FIG. 10 is a graph of the dilution curves of Example XIII.

It has been found that for an early diagnosis of cancer by measuring tumor markers, although preferably separate measurements are made on the nature of the tumor marker f and and their amounts in practice, it is possible to base the diagnosis on the total amount of tumor markers as can be seen from Examples II, IV, VI and XI below. Based on this discovery, further work has been carried out on a method which makes it possible to measure the amount of two or more substances in one sample to be measured simultaneously in the form of a total amount with a single measuring treatment. As a result, it was found that the total amount of two or more substances to be measured can be measured by a single measurement treatment based on an immunological measurement principle, the measurement being performed by binding of two or more different antibodies, correspondingly with two or more antigens to be measured, on the same insoluble carrier, or by mixing and using two or more insolubilized antibodies, obtained by binding antibodies, corresponding to two or more different antigens to be measured, to separate onoptoshare carriers, without some change in the reactivity between each antigen and corresponding antibody is caused. This is the invention.

8302708 t t -3-

The immunological measuring methods that form the basis of the present measuring method are widely used as methods for measuring physiologically active substances present in extremely small amounts in samples such as serum, urine, etc., for example the concentrations of peptide hormones, steroids, proteins, etc., the concentrations of drugs administered, etc. Of these reactions, the hemagglutination reaction and the latex agglutination reaction are most preferably used because they are easily and in a shorter time feasible, while also enzyme immunoassays, radioimmunoassays and fluorescence immunoassays have proven beneficial because of their sensitivity and quantitative results.

The principles of the measurement methods are summarized below: (1) Agglutination reaction: When an unknown amount of antigen to be measured reacts with a corresponding antibody bound to a particulate carrier (referred to below as solid phase), such as red blood cells and a polymer latex, binds the antigen to the antibody on the solid phase in proportion to the amount present, whereby the solid phase agglutinates. The extent of this agglutination is measured and the amount of antigen to be measured is determined by comparison with the degree of agglutination achieved when the same substance is measured at a known concentration.

(2) Sandwich method; When an unknown amount of an undetected antigen (antigen to be measured) is reacted with a corresponding antibody bound to a solid phase (first reaction) 9, this antigen binds to the antibody to form an antigen / antibody complex. When a given amount of the same antibody, labeled with a label, is reacted with this antigen / antibody complex (second reaction), the labeled antibody is bound to said complex, but part of the labeled antibody that was compared in excess with the binding capacity of the complex, does not bind to it and thus remains in free form.

Then, the solid phase is separated from the liquid phase, the activity of the marker in the solid phase or in the liquid phase is measured and the amount of undetected antigen to be measured is determined on the basis of a standard curve obtained by similar operations with undetected antigen at known concentrations.

8302708 \ t -4- (3) Competitive reaction method: When an unknown amount of an undetected antigen (antigen to be measured) and a given amount of a labeled antigen are reacted competitively with a corresponding antibody bound on a solid phase, then bond- Labeled antigen and labeled antigen, respectively, to this antibody proportional to their respective amounts, so that the amount of labeled antigen that binds to the antibody is inversely proportional to the amount of unlabeled antigen. Then, the solid phase is separated from the liquid phase and the activity of the marker in the solid phase or in the liquid phase is measured and the amount of antigen determined on the basis of a standard curve obtained by performing similar operations with unlabelled substances at known concentrations.

(4) Immunometric method: When an unknown amount of an undetected antigen (antigen to be measured) is reacted with a given amount of a corresponding labeled antibody and then the same antigen bound to a solid phase is added to react with unreacted labeled antibody. , the labeled antibody will bind to the antigen on the solid phase, inversely proportional to the amount of unlabeled antigen present. Then, the solid phase is separated from the liquid phase and the activity of the marker in the solid phase or in the liquid phase is measured and the amount of undetected antigen determined on the basis of a standard curve obtained by performing similar operations with the unlabelled substance in known concentrations.

Thus, the invention relates to a method and a reagent for simultaneously measuring the total amount of two or more substances to be measured contained in one sample using the principles of the immunological measuring methods described above.

The present measuring method can be applied to any of the usual immunological measuring methods in which an antibody or antigen is bound to an insoluble support, for example the hemagglutination reaction, the agglutination inhibition reaction, the competitive reaction method, the sandwich method, the immunometric method, etc.

The present method is now schematically described by means of the agglutination reaction and the sandwich method.

8302708 * - i -5- (1) Agglutination reaction: When a sample that is expected to contain the four substances A, B, C and D must be measured ^ four different antibodies corresponding to the substances to be measured namely anti-A antibody, anti-B antibody, anti-C antibody and anti-D antibody bound to the same insoluble particulate support, or they are bound separately on separate insoluble particulate supports to prepare a suspension of four different suspensions of insoluble particulate supports with these four antibodies. when the sample is reacted with these suspensions, the substances A, B, C and O to be measured bind with the corresponding antibodies on the supports, respectively, whereby agglutination of the particulate supports occurs.

By measuring the degree of agglutination, the total amount of the two or more substances to be measured can be measured in one sample.

(2.) Sandwich method r When a sample believed to contain four substances A, B, C and D must be measured ^ four different antibodies corresponding to the substances to be measured, namely anti-A antibody, anti-B antibody , anti-C antibody and anti-D antibody bound on the same insoluble support or bound separately on separate insoluble supports to yield an insoluble support or four different insoluble supports containing these four antibodies. When the sample comes into contact with these insoluble carriers, the substances A, B, C and D to be measured, respectively, will bind to the corresponding antibodies on the carriers. After separation of the solid phase, if necessary, labeled antibodies are obtained by binding a label to anti-A antibody, anti-B antibody, anti-C antibody and anti-D antibody, respectively, with the solid phase reacted, the respective labeled antibodies bind to the substances to be measured, respectively. The solid phase is then separated and the total activity of the label bound to the solid phase is measured. Thus, the total amount of two or more substances to be measured in a sample can be measured simultaneously.

The insoluble particulate support in the present agglutinatium method can be any conventional material. That is, cells of e.g. bacteria, red blood cells, etc., organic polymer- 8302708% i.

Latices, such as a polystyrene latex, a polystyrene latex with carboxyl groups, a styrene divinyl benzene copolymer latex, a styrene divinyl benzene polymer latex with hydroxyl or carboxyl groups, a poly vinyl alcohol latex, a vinyl acetate, a vinyl acetate. an inorganic material, such as silica, carbon black, alumina, etc., either alone or in some combination.

As the material for the insoluble support in the sandwich method, the competitive method and the immunometric method according to the invention, any usual material for this can be used, for example polystyrene, polyethylene, polyacrylic, teflon, paper, glass, agarose, etc. each on its own or in any desired combination. Furthermore, any desired shape can be used, for example spheres, rods, discs or a container, for example an optical cell, a test tube, etc., although any other shape can also be used.

The antibodies used in the invention can be the common multi-clone antibodies, although use of monoclonal antibodies still yields better results. For example, monoclonal antibodies result in an improvement in the sensitivity and accuracy of the measurement, a decrease in the reaction time, an improvement in the specificity, simplification of the measurement treatments, exclusion of non-specific reactions, exclusion of inhibition of the reaction by body components. in the sample, for example, serum, urine, etc., and the like. When monoclonal antibodies are used in the agglutination reaction, it is necessary to use two or more monoclonal antibodies corresponding to different antigen sites of one substance to be measured in combination to achieve agglutination.

As a method of binding one or more antibodies or antigens to an insoluble carrier, the method of Clinica 30 Chimica Acta, 48: 15 (1973), Journal of Immunology, 116: 1554 (1976) of Science, 158: 1570 (1967 ) are applied. When two or more different antibodies are to be bound to a single insoluble support, an antibody solution can be mixed by mixing the antibodies to be bound in a fixed ratio, for example, a solution with 0.5 mg / cm 2 anti-APP antibody, 0.1 mg / cm ^ anti-HCG antibody and 0.25 mg / cm ^ anti-CEA antibody in 0.05 M with phosphate 8302 7 Ö8> * -7- buffered saline with pH 6.4, then prepare the mixed antibody solution are brought into contact with the insoluble carriers and the reaction is carried out at 30-56 ° C for 2-3 hours. After this reaction, the carriers are washed with a physiological saline solution to yield carrier-bound antibody.

When anti-AFP antibody, anti-HCG antibody and anti-CEA

antibody can be bound separately to individual insoluble

carriers, anti-AFP antibody, anti-HCG antibody and anti-CEA

Antibody ^ individually dissolved in a 0.05 M phosphate buffer-containing physiological saline with pH 6.4 in concentrations of 0.5 mg / cm ^, 3 3 0.1 mg / cm and 0.25 mg / cm ^ in contacted with individual insoluble carriers and each mixture reacted at 30-56 ° C for 2-3 hours. After the reaction, the insoluble carriers are washed with physiological saline and thus the respective antibody-containing carriers are obtained. The mixing ratio of the insoluble antibodies thus obtained is preferably selected within the range 1-10: 1-10: 1-10, although this may vary depending on the expected amount of antibody to be bound and the titer of each antibody.

The amount of each antibody to be bound to the insoluble support is preferably 10-0.05 mg / cm ^, most preferably 5-0.1 mg / cm ^, although this also varies depending on the substance to be measured, the amount of each substance to be measured in the sample and the titer of each antibody. When the measurement is performed, the optimal conditions for the experiment are determined as: "Determined, since the amount of sample to be used, the amount of each labeled antibody or tagged antigen, and the conditions, such as reaction time, temperature, etc., vary depending on the nature of each substance to be measured, the titer of each antibody used, and the nature of the markers.

The production of the labeled antibody or labeled antigen can proceed according to a conventional procedure.

If the method according to the invention is carried out on the basis of the competitive reaction, the labeled anti-35 to be used is not that which binds competitively with the substance to be measured to the undissolved antibody, so that generally the same substance as the 8302708 1 * -8- substance to be measured is used as antigen. However, the physiologically active substances present in the human body can occur bound to other body components or undergo partial metabolism, sometimes slightly different from the form in which.

5 they are present outside the human body. In such a case, a substance which has substantially the same reactivity from an immunological standpoint can also be used as an identical substance with the substance to be measured.

Although the invention aims to measure two or more substances simultaneously, it is also possible to measure a single substance using only one labeled antibody, even when two or more insolubilized antibodies are used, unless the substances to be measured are each other to disturb.

As the intermediates, enzymes (e.g., peroxidase, 15β-galactosidase, alkaline phosphatase, glucose oxidase, etc.), radioactive isotopes, such as J, H, etc., may include fluorescent substances, such as fluorescein isothiocyanate, tetramethylrodamine isothiocyanate, etc. are applied.

In the agglutination reaction, the total amount of two or more substances to be measured can be measured simultaneously by using a carrier with two or more antibodies. In the sandwich method and the competitive reaction method, by using two or more labeled antigens or labeled antibodies corresponding to two or more antibodies attached to the insoluble carriers, the total amount of the two or more substances to be measured can be measured simultaneously. Furthermore, in the sandwich method and competitive reaction method, using a single labeled antigen or labeled antibody can also measure a single substance. For example, a sample diluted to an appropriate concentration is added to a test tube to which an antibody is bound, after which the antigen-antibody reaction is performed. After this reaction is complete, the test tube is washed with distilled water, a labeled antibody is added and a reaction is again performed.

Then, after washing with distilled water, the activity of the solid phase marker (test tube) is measured appropriately. From the measured values obtained, the amount of substance present in the sample can be calculated based on a standard curve obtained by equal operations with the standard substance at known concentrations. The standard substance in this case can be either a single antigen or a mixture of a series of antigens.

The measuring method according to the invention can measure any substance that can also be measured according to the usual immunological measuring method. Of particular interest as substances to be measured are, for example, tumor markers which are of great importance in the early diagnosis of tumors, the assessment of a theurapeutic effect, etc.

Examples include carcino embryo antigen (CEA), α-fetoprotein (AFP), human chorionic gonadotropin (HCG), 8 ^ microglobulbulin (S ^), basic fetoprotein (BFP), alkaline phosphatase (ALP), γ -glutamyl transpeptidase (γ-GTP), pregnancy Sj-glycoprotein (SP ^), pregnancy glycoprotein (SP ^), immunosuppressive acid protein (IAP), immunosuppressive alpha-macroglobulin, acid iso-ferritin, fibrinogen, haptoglobin, calcitonin, steroid hormones, polyamines, DNA binding proteins, alpha-antitrypsin, pancreatic cophaleal antigen, galactosyl transferase (GT-IT), etc .; as well as various substances, which are currently still under review. Also substances with an interest in the diagnosis and prophylaxis of hepatitis due to an infection with hepatitis virus after a blood transfusion? these are hepatitis B virus antigens (HBs, HCc, HBe) and non-A, non-B hepatitis virus antigens.

Examples of combinations of two or more different substances to be measured include (1) AFP, CEA, HCG, (2) SP ^, fibrinogen, (3) AFP, CEA, HCG, SP ^, fibrinogen, (4) fibrinogen, haptoglobin , ferritin, (5) haptoglobin, α-immunosuppressive α2-macroglobulin, α-antitrypsin, (6) ALP, γ-GTP, GT-II, (7) polyamines, steroid hormones, (8) HBs, HBc, HBe and the like.

The invention makes it possible to shorten the measuring time, because otherwise, when measuring a number of antigens, antigens to be measured separately according to a usual method had to be measured separately and more time was required for this. Furthermore, the amount of sample required can be significantly reduced.

The invention is illustrated by the following examples.

35 8302708 V l '' -10-

Example I

Measurement of the total amount of fibrinogen, haptoglobin and ferritin a) Preparation of a latex reagent with bound antibody.

Anti-fibrinogen antibody (DAKO Co.), anti-haptoglobin 5 antibody (DAKO Co.) and anti-ferritin antibody (DAKO Co.) were diluted and in a pH 6.4 (0.05 M phosphate buffered saline) solution. PBS) mixed to concentrations of 5 3/3, 5 mg / cm, 1.8 mg / em and 0.7 mg / cm, respectively. 0.5 cm 10% polystyrene latex slurry (uniform latex particles from Dow Chemical Co.) was added to 2 cm of the above mixed antibody solution and reacted at 37 ° C for 2 hours with stirring. At the conclusion of the reaction, the reaction mixture was cooled with ice, centrifuged, washed with PBS and suspended in PBS with 1% bovine serum albumin (BSA) to a bound antibody-containing latex reagent.

B) Preparation of standard solutions.

Fibrinogen (Sigma Co.), haptoglobin (Sigma Co.) and ferritin (Sigma Co.) were separately dissolved in PBS containing 1% BSA to concentrations of 120, 60, 30, 15 and 0 mg / 1, 12, 6, respectively. , 3, 1.5 and 0 mg / 1 and 0.8, 0.4, 0.2, 0.1 and 0 mg / 1.

C) Measurement of fibrinogen, haptoglobin and ferritin.

3 20 mm of each of the solutions of fibrinogen, haptoglobin and ferritin at the concentrations according to b) were placed on a 3 slide, then 50 mm PBS containing 1% BSA and 20 mm of antibody-containing latex reagent according to a) were added. The mixture was reacted with stirring for 3 minutes and the degree of agglutination observed microscopically. The reaction without agglutination was indicated as negative (-) and that with agglutination was indicated as positive (+, ++ or +++ as the strength) giving four signs. The sensitivity of the reagent to fibrinogen, hapto-globin and ferritin was 15 mg / 1, 1.5 mg / 1 and 0.1 mg / 1, respectively.

Example II

Measurements on patient serum samples

Serum samples were obtained from 18 liver cancer-35 patients, 20 gastric cancer patients, 20 patients with colon cancer and 8 lung cancer patients, 25 patients with various benign diseases and 20 healthy people, the measurements being performed according to the invention. The procedure for these measurements was the same as that of Example Ic. The results are shown in Table A.

5 TABLE A

Number of Assessment

Disease patients - + ++ +++% Positive liver cancer 18 1 2 9 6 94 stomach cancer 20 3 4 8 5 85 10 colon cancer 20 2 3 7 8 90 lung cancer 8 0 242 100 benign diseases 25 19 5 1 0 24 healthy people 20 18200 10

This Table A shows that an arc percentage of cancer patients was rated positive, demonstrating the utility of the present method for screening cancer patients.

Example III-

Measurement of the total amount of fibrinogen, haptoglobin and ferritin 20 a) Production of anti-fibrinogen antibody-containing latex.

Anti-fibrinogen antibody (DAKQ Go.) Was diluted with PBS

3 3 to a concentration of 5 mg / cm. 0.5 cm 2 of a 10% polystyrene latex slurry (mean particle diameter 0.48 µl, Dow Chemical) was added to 2 cm of this dilute solution and the mixture was stirred for 2 hours at 37 ° C. At the conclusion of the reaction, the reaction mixture was cooled with ice, centrifuged, washed with PBS and then suspended in PBS with 1% BSA to form previously bound anti-fibrinogen antibody-containing latex.

b) Production of anti-haptoglobin antibody on latex particles.

Anti-haptoglobin antibody was diluted with PBS to a concentration of 1.8 mg / cm 2 and this solution was converted into a latex with anti-haptoglobin antibody with a 10% polystyrene latex suspension according to a).

8302708 I) -12- c) Production of anti-ferritin antibody on latex.

Anti-ferritin antibody (DAKO Co.) was diluted with PBS to 1.2 mg / cm 2 and this solution with a suspension of 10% polystyrene latex with carboxyl groups (average particle diameter 0.25 µm, 5 Dow Chemical) according to a) converted into a latex with anti-ferritin antibodies thereon.

d) Production of antibody on latex reagents.

The latex with anti-fibrinogen antibody, the latex with anti-haptoglobin antibody and the latex with anti-ferritin antibodies 10 were mixed in a 2: 5: 1 ratio to form a latex reagent with antibodies.

e) Measurement of fibrinogen, haptoglobin and ferritin.

3 20 mm of each of the standard solutions of fibrinogen, haptoglobin and ferritin produced according to example X b) were applied to a slide, then 50 mm PBS containing 1% BSA and 3

Add 2Q mm of the latex reagent of d) above to the standard solution on the glass slide. This mixture was allowed to react with stirring for 3 minutes, after which the degree of agglutination was observed under a microscope. A reaction without agglutination was indicated as negative (-), a reaction with an agglutination was indicated as positive (+, ++ or +++ depending on the degree of agglutination). The sensitivity of this reagent to fibrinogen, haptoglobin and ferritin was 15 mg / 1, 1.5 mg / 1 and 0.1 mg / 1, respectively. Example IV

25 Measurements on patient serum samples

Serum samples obtained from 15 liver cancer patients, 20 gastric cancer patients, 17 patients with colon cancer, 12 lung cancer patients, 25 patients with various benign diseases and 20 healthy people were measured using the reagent of Example III. The results are shown in Table B.

8302 70S

-13-TABLE · B

Number of Rating (%)

Diseases patients + ++ +++ positive liver cancer 15 1275 93 stomach cancer 20 3485 85 colon cancer 17 2267 88 lung cancer 12 1263 92 benign diseases 25 20 4 1 0 20 healthy people 20 19 100 5

As shown in Table B, cancer patients were highly positive, indicating that the present method of measurement is particularly suitable for screening cancer patients.

Example V

5 Production of purified AFP and anti-AFP antibodies

a) Production of purified AFP

16.2 g of crude AFP extract was obtained from 5 liters of ascites of liver cancer patients by salting out with ammonium sulfate. The crude extract was purified by affinity chromatography using 50 cm rabbit anti-AFP antibodies bound to 3 Sepharose 4B (1 mg antibody / cm Sepharose) to obtain 924 mg purified AFP.

b) Production of anti-AFP monoclonal antibodies 50 µg of the purified AFP was administered subcutaneously along with Freund's complete adjuvants (FCA) to female BALB / c mice.

The administrations were repeated four times at one week intervals and four days after the last administration the mildly isolated and these mild cells collected. The mild cells were washed with Dulbecco modified g MEM medium (D-MEM below), 1 x 10 cells were counted and mixed with 1 x 10 4 cells in mouse myeloma cells (P3-NSI / 1-Ag 4-1).

3

This was subjected to a cell fusion in 1 cm D-MEM containing 42.5% polyethylene glycol 1540 and 7.5% dimethyl sulfoxide for 1 minute at 37 ° C. To these cells, HAT medium (RPMI-1640 containing 8302708-14 containing hypoxantine, aminopterin, tymidine and 10% bovine fetal serum) was added 3 to a volume of 20 cm, after which the cell mixture in amounts of 0.2 cm each microplate was applied with 96 wells and cultured for 2 weeks. After this, the antibody activity in the culture supernatant was measured in the wells. After this, the cells were collected in the activity wells and added

added to 40 cm RPMI-1640 medium containing 10% bovine fetal serum and tymus cells from BALB / c mice. This cell suspension was again divided into two 96-well microplates and grown for 1 week to 9 clones of 10 anti-AFP antibody-producing hybridomas. These were transferred. to cultures. technical scale and 1 liter of each supernatant culture liquid obtained subjected to an affinity chromatography using 50 cm purified AFP-containing sepharose 4B

3 (0.5 mg AFP / cm Sepharose) after which 4.2-11.6 mg monoclonal antibodies were obtained, respectively. The respective antibodies were designated as batches 1-9.

c) Identification of antigen recognition sites. Production of anti-AFP antibody-containing test tubes. Portions of 1 cm PBS containing 0.5 mg of anti-AFP 20 monoclonal antibody from batches 1-9 were added to individual polystyrene test tubes that had been washed with PBS, after which each test tube was incubated at 37 ° C for 3 hours to adhere the antibody to the inner wall of the test tube. After the reaction, the test tubes were washed with PBS to obtain monoclonal antibody-containing test tubes.

ii) Production of enzyme-labeled anti-AFP antibody 5 mg. horseradish peroxidase (Behringer Manheim Co.), grade I, referred to below as HRPO> was dissolved in 1.0 cm of a 3 0.3 M sodium bicarbonate buffer and 0.1 cm 1% 1-fluoro-30 2 to this solution , 4-dinitrobenzene in ethanol was added and the mixture was reacted for 3 hours. Further, 1.0 cm 2 of a 0.06 M sodium periodate solution was added to this mixture, the reaction was allowed to proceed for 30 minutes, then an additional 1.0 cm 2 of a 0.16 M ethylene glycol solution was added and the reaction Continued for 1 hour. The reaction mixture was then dialialized against 0.01 M sodium carbonate solution with pH 9.5. To the dialyzed solution, 5 mg of each of the 8302708-15 batches of anti-AFP antibody of b) was added and each mixture incubated at room temperature for 3 hours. Then 5 mg of sodium borohydride was added and the reaction continued overnight. The reaction mixtures were dialyzed against 0.01 M 5 PBS at pH 7.2, respectively, to obtain a number of HRPO-labeled anti-AFP antibodies.

iii) Identification of antigen recognition sites

To each of the test tubes containing anti-AFP antibodies, obtained according to i), 0.1 cm of the standard solution was added, 10 diluted with PBS to 100 ng / cm3 AFP according to a) and 0.4 cm3 of a 100-fold dilution of each HRPO-labeled anti-AFP antibody according to ii) after which each reaction was performed for 30 minutes. After the reaction was complete, each test tube was washed with a washing solution, 3 0.5 cm of an enzyme substrate solution containing 20 mg / dl asm 15 O-phenylenediamine and 6 mM hydrogen peroxide, and this reaction was also carried out for 30 minutes. 2 ml of 1 N hydrochloric acid was added to stop the enzyme reaction and the absorbance of the reaction mixture measured at 492 nm. The results are shown in Table C where + denotes the combinations with a color reaction and - the combinations without a color reaction.

TABLE · C

HRPO-labeled antibody I 123456789 3 µl - + - + - + +

• H

• h2 - - - + - + - + +

+ J

25 <3 3— - - + - + - + + $ 4 + + + - + + + -

Rj S5 --- + - + - + + <u S? 6+ + + + + - + + + <e

Yes7 --- + - + - + + approx. 30 ° 8 + + + - + + + - 0 «§9 + + + - + + + - o

Based on the differences in the antigen recognition sites, the nine batches of monoclonal anti-AFP antibodies were classified into three groups, namely a first group of batches 1, 2, 35, 5 and 7, a second group from batch 6 and a third group from the 83027C8-16 batches 4, 8 and 9. These groups were designated: anti-AFP antibodies (A), (B) and (C). Of these, (A) will be used as gel-solubilized antibody and (C) as labeled antibody in the following examples.

Example VI

Production of purified CEA and anti-CEA. antibodies

a) Production of purified CEA

40 g of colon cancer tissue was chopped and homogenized after an addition of 100 cm of distilled water in a homogenizer. An equal volume of 1.2 M perchloric acid was added to this suspension and this mixture was extracted with stirring for 30 minutes. The centrifuged supernatant was dialyzed against distilled water to obtain crude CEA extract.

* 3

This crude extract was concentrated to 10 cm and gel-filtered with Sepharose 4B, equilibrated with physiological saline and collected the first protein fraction. This fraction was then gel-filtered again using Sephadex G-200, equilibrated accordingly and collected a two protein fraction, which was then concentrated to 2 cm 2 to obtain 135 µg of purified CEA.

b) Production of anti-CEA monoclonal antibodies

Eight clones of anti-CEA antibody-producing hybridomas were obtained from the purified CEA of a) by a procedure according to Example Vb. For the immunization of the mice, 30 µg of purified CEA was used for each administration.

1 x 106 hybridoora cells were inoculated intraperitoneally into a female BALB / c mouse administered intraperitoneally 0.5 ml pristan (2, 6, 10, 14-tetramethylpentadecane from Wako Pure Chemicals, Co., Ltd.). The ascites was collected two weeks later.

Each ascites was chromatographed on DEAE cellulose, equilibrated with a 0.01 M phosphate buffer at pH 7.0, thus obtaining a monoclonal anti-CEA antibody in the unabsorbed fraction. As a result of the identification test on the antigen recognition sites of Example Vc, the respective antibodies were classified into three groups of 5 batches, 2 batches and 1 batch each, which were designated as anti-CEA antibody (A), (B) and (C). Of these, 8302708-17 anti-CEA antibody (A) will be used as an insolubilized antibody and anti-CEA antibody (B) as labeled antibody in the following examples.

Example VII

Preparation of HCG-8 and anti-HCG-6 antibodies a) Production of HCG-8 subunits 1 g HCG (2000 IU / mg) was dissolved in 2 cm ^ 0.025 M phosphate buffer with pH 5.6 and chromatographed on 3 g DEAE-sefadex A-50, equilibrated with the same buffer. The fraction eluted with 0.05 M phosphate buffer of pH 5.6 was collected and dialyzed against distilled water until 308 mg of purified HCG was obtained, which was then lyophilized. 300 mg of this purified HCG was dissolved in 10 cm ^ 10 M urea solution with pH 4.5 and a reaction of 1 h at 40 ° C was performed therewith, followed by chromatography on 2 g DEAE-sefadex A-50, equilibrated with a solution containing 0.03 M glycine and 10 M urea. The fraction, eluted with a solution containing 0.2 M glycine, 1 M NaCl and 8 M urea, was dialyzed against physiological saline to obtain 147 mg HCG-6 subunits.

B) Production of anti-HCG-S antibodies

Eleven batches of anti-HCG-6 antibody-producing bybridomas were obtained from the HCG-8 units produced under a) by a similar procedure as described in Example Vb. In the antibodies obtained from these batches, the identification of the 25 antigen recognition sites was performed according to example Vc and the products were divided into two groups, one of 8 batches and one of 2 batches, respectively designated as anti-HCG-B antibodies (A) and B). The anti-HCG-8 antibody (A) will be used as an insolubilized antibody and (B) as a labeled antibody in the following examples. When these antibodies were tested for their cross-reaction with lutenylating hormone (LH) using the above combination, the reactivity was found to be less than 1% when the reactivity to HCG was set to 100%.

8302708 -18-

Example VIII

Measurement of the total amount of & ΓΡ, CEA and HCG

a) Production of antibody on test tube reagent

To polystyrene test tubes 1 cm3 of a solution containing 3 3 5 1 mg / cm monoclonal anti-APP antibody / 0.5 mg / cm monoclonal anti-CEA antibodies and 0.25 mg / cm monoclonal anti-HCG antibodies was added (See Examples V, VI and Vlij added and then each test tube incubated for 3 hours at 37 ° C. After this, each test tube was washed with PBS to yield reagent-containing test tubes.

b) Preparation of standard solutions

The APP according to example Va ,, the CEA according to example Vla and HCG were diluted with PBS containing 1% BSA with solutions of 3 3 80, 40, 20, 10, 5 and 0 ng / cm, 80, 40, 20, 10 .5 and 0 ng / cm and 80, 40, 20, 10, 5 and 0 miu / cm3, respectively, were obtained.

c) Measurement of APP, CEA and HCG

To the reagent-containing test tubes with antibody obtained according to a), 0.1 cm 3 portions of APP, CEA and HCG standard solutions of the respective concentrations were added, followed by an addition of 0.4 cm portions of PBS containing 1% BSA, after which each time the reaction was carried out at room temperature for 2 hours. After the reaction was completed, the test tubes were washed with distilled water. Separately, enzyme-labeled antibodies obtained according to Examples V, VI and VI1 were diluted and mixed with PBS containing 1% BSA into solutions containing a 2000-fold dilution of enzyme-labeled anti-APP antibodies to an 800-fold dilution of enzyme-labeled anti-CEA antibody and a 500-fold dilution of enzyme-labeled anti-HCG antibody. 0.5 ml aliquots of this mixture were added to the test tubes shown above and the reaction was carried out at room temperature for 2 hours each time. At the conclusion of the reaction, the test tubes were washed with distilled water, 3 0.5 cm portions of a substrate solution (6 mM / 1 hydrogen peroxide and 20 mM / 1 o-phenylenediamine in PBS) were added, and the reaction each time with the exclusion of light. minutes. After this, 2 ml of 1N hydrochloric acid was added to each tube to stop the reaction and the absorbance of the reaction mixture at 492 nm was measured. For comparison, measurements were also made for the cases where the labeled antibodies were not mixed but reacted separately. The results are listed in Figures 1 to 4. The combinations of the insolubilized antibodies and the labeled antibodies used in the respective Figures are shown in Table D.

TABLE D

Insolubilized Labeled Antibody Antibody anti-AFP Antibody, anti-CEA Antibody and Figure 1 anti-HCG Antibody ^. __. .... in the mixture anti-AFP antibody anti-CEA antibody and. . __. . .

anti-AFP antibody anti-HCG antibody only in the mixture figure 3 anti-CEA antibody only figure 4 anti-HCG antibody only ----

The results show that since AFP, CEA and HCG do not cross-react, they can be individually measured using the respective labeled antibodies, even if these substances are present in the mixture} and that even when the labeled antibodies are in the mixture are used, only the respective corresponding antigens and antibodies react with each other and this reactivity is not affected by the presence of other substances.

Example IX

15 Measurements on patient samples

Serum samples from 10 liver cancer patients, 10 gastric cancer patients, 10 colon cancer patients, 10 patients with benign diseases and 10 healthy people were measured by their total amount of AFP, CEA and HCG, respectively. Each measurement was made with 0.1 cm 2 of either the standard solution or the serum to be tested according to the procedure of Example VIIIc. The results are from 8302708

-% X

-20- printed by the direct absorbance value at 492 nm and also by the CEA conversion value obtained by using this absorbance value in the CEA standard curve. For comparison, AFP, CEA and HCG were also measured separately in the same samples. The results are shown in Table E.

8302708 -2t-

TABLE E

Invention Comparison

Disease Absorption CEA environment

at AFP put CEA HCG

• 492 nm worth ^ ng / απ ng / cm miu / ca ng / cm 0.570 20.0 20.1 2.1 2.0 0.392 10.7 10.5 5.3 1.8 1.608 85.2 101.8 1.1 3.1 0.231 4.0 2 »3 2» 2 2 »°

Liver 3,205 147.5 158.7 0.8 2.0 Cancer 0 <261 5.3 0.2 0.3 4.4 1.785 99.7 60.1 51.1 3.5 0.431 10.2 12. 3 ^ ·} 2 2 * 7 0.818 32.0 33.9 0.8 1.3 0.211 57.1 63.2 0.5 1.5 0.473 15.0 - 4.0 8.8 4.1 0.915 27 .5 1.7 0.7 30.1 0.731 28.8 1.5 1.3 30.8 0.321 7.1 3.1 4.1 0.9

Gastric 0.492 19.8 1.1 1.7 21.1 Cancer 0501 16.8 1.0 17.2 0.8 0.481 18.1 16.3 0.3 4.1 0.336 8.1 4. 4 1.1 3.1 ji 0.270 5.1 1.0 2.2 3.7 jifi 0.260 4.7 2.0 1.0 4.0 j 8302708

Invention Comparison

TABLE S

(continuation)

V

-22-

Disease Absorption CEA environment

at AFP put CEA HCG

• 492 nm worth ^ ng / cm ng / cin miu / cm ng / am 0.788 4.4 3.2 0.8 2.1 3,109 138.5 1.5 152.1 2.1 1.121 50.3 4.0 50.5 3.3 '. 0.228 3.8 1.0 2.8 0.9 thick. colorectal cancer ° '388 10'Χ l 0.8 0.470 14.8 1.2 13.0 3.2 0.323 6.9 8.8 0.7 1.4 0.450 13.9 10.3 6.1 1.5 2,712 119.3 138.1 1.5 1.8 0.620 20.9. 2.5 21.8 2.0 0.231 4 .0 1.3 1.3 0.3 3.0 0.219 3.8 1.3 0.2 1.9 0.320 6.9 | 3.1 3.2 2.2 0.301 6.0 | 2.1 3.1 2.5 benign. 0.371 9.8 y 2.2 6.1 1.1 diseases! 0.333 - 8.5 y 2.5 1.8 3.5 0.261 4.9 | 0.8 2.1 3.3 i 0.321 7.1! 2.9 2.2 2.2 0.271 5.1 0.8 3.1 2.8 0.270 5.1 4.1 0.9 1.0 8302708

TABLE E

(continuation)

Invention Comparison -23-

Disease Absorption GEA-converted AFP ^ CEA ECG ^ • 492 nm worth | ng / cm ng / cm mln / cm ng / cm f 0.230 4.0 1.1 1.2 2.2 0.241 4.2 1.2 1.3 2.2 0.281 5.3 3.1 1.5 3 .2 healthy 0.267 4.8 0.7 1.5 3.8 people _ _ 0.259 4.7 0.7 2.8 3.3 0.268 '4.8 2.0 2.1 2.0 0.221 3.7 1.1 1.5 1.8 0.260 4.7 0.8 3.1 1.7 0.253 4.5 0.7 2.1 2.1 0.231 4.0 0.7 0.9 2.1

Table F below shows the percentage of patients' positives of each disease obtained with the individual measurements, provided that the diagnostic default values for suspected cancer cases are 10 ng / cm3 higher for AFP, 5 ng / cm3 or higher 5 for CEA and 5 miu / cm3 or higher for HCG. Table F also includes the percentages of patient positives for each disease in the measurements of Table E, where when measuring the total amount of the three substances of the invention, an absorbance at 492 nm of 0.350 or greater or its converted CEA value of 8.5 ng / cm3 or higher 10 is considered positive.

8302708 r

-24-TABLE F

Disease Invention AFP CEA HCG One oJ ™ er markers liver cancer 80% 80% 20% 0% 80% stomach cancer 60 10 20 30 60 v 60 10 60 0 60 colon cancer benign 10 0 10 0 10 diseases healthy q 0 0 0 0 people

Furthermore, when calculating the percentages of positive in the patients of each disease, assuming that a patient is cancer positive, if at least one of the three tumor markers individually exceeded the default value, these percentages were positive, which are also in the right column of Table F included the same as the percentages of cancer positive in the case where the three tumor markers were measured in their total amount (the second column of Table F). This shows that in a diagnosis of cancer it is not necessary to measure the tumor labels individually, but that in practice a diagnosis is possible from a measured value of the total amount together.

Example X.

Measurement of the total amount of AFP, CEA and HCG a) Production of polystyrene beads with antibodies thereon Monoclonal anti-AFP antibodies, anti-CEA antibodies and anti-HCG antibodies prepared according to Examples V, VI and VII were diluted to concentrations of 1 , 0.8, and 0.5 mg / cm. 2.5 mm diameter polystyrene beads were dipped into each solution and each mixture left at 37 ° C for 3 hours. At the end of the reaction, the 20 beads were washed with PBS to yield antibody-containing polystyrene beads, and a reagent was built up by combining one bead from each batch to three.

8302708 -25-

b) Measurement of AFP, CEA and HCG

To test tubes with an inner diameter of 10 mm and a length of 60 mm 0.1 ml portions of AFP, CEA and HCG standard solutions with the concentrations according to example VUIb were added, 5 and 0.4 cm portions of a PBS solution containing 1% BSA and three different antibody-containing polystyrene beads added thereto. After stirring, the reaction was allowed to proceed at room temperature for 2 hours. At the end of the reaction, the polystyrene beads were washed with distilled water. Separately, the enzyme-labeled antibodies of Examples 5, 6 and 7 were diluted and mixed in a PBS containing 1% BSA to solutions with a 2500-fold dilution of enzyme-labeled anti-AFP antibodies, a 1000-fold dilution of enzyme-labeled anti-CEA antibody and a 650-fold dilution of enzyme-labeled anti-HCG antibody. 0.5 cm portions of each mixture were added to these beads and the reaction was carried out at room temperature for 2 hours each. At the end of the reaction 3, the polystyrene beads were washed with distilled water, 0.5 cm portions of a substrate solution of 6 mM / l hydrogen peroxide and 20 mM / 1 o-phenylenediamine in PBS) were added and each mixture was protected at room temperature for 30 minutes. against the light, converted. After this, 2 cm ^ 1 N hydrochloric acid was added each time to stop the reaction.

The absorbance was measured at 492 nm. For comparison, measurements were made for the cases where the labeled antibodies were not mixed but reacted separately. The results are listed in Figures 5-8. The combinations of the insolubilized antibodies and the labeled antibodies used in the respective figures are shown in Table G.

8302708 -26-

TABLE G

Insolubilized Labeled Antibody Antibody anti-AFP Antibody, anti-CEA Antibody and anti-HCG Antibody

Mixture of 3 beads in the mixture (anti-AFP antibody ^..,, I anti-AFP antibody on grain ƒ anti-CEA antibody> aa on grain and 'anti-HCG antibody anti-CEA antibody on grain J only fiomir's anti-HCG antibody. ^ only

The above results, like Example VIII, show that AFP, CEA and HCG do not show any cross-reactions, and can be measured separately using the respective labeled antibodies, even when these substances are present in a mixture, 5 even if the labeled antibodies are present in a mixture, only the respective corresponding antigens and antibodies react and this reactivity is not affected by the presence of other substances.

Example XI

10 Measurements on patient serum samples

Serum samples taken from 10 liver cancer patients, 10 gastric cancer patients, 10 colon cancer patients, 10 patients with benign diseases and healthy people, respectively, were measured on their total amount of AFP, CEA and HCG. Each measurement was made using 0.1 cm 3 of either a standard solution or the serum to be tested according to methods according to Example VIIIc. The results are expressed via the direct absorbance value at 492 nm and also by the CEA conversion value obtained by applying this absorbance value to the CEA standard curve. For comparison, AFP, CEA 20 and HCG were also measured separately in the same samples. The results are shown in Table H.

8302708 -27- TABLE · Η

Invention Comparison

Disease Absorption CEA-environment. at AFP ^ CEA 2 ®-G 3 492 nm worth ^ ng / cm ng / cm miu / cm ng / cm 1.55 <80 <320.5 9.6 1.2 t 1.55 <80 <j173, 6 3.0 1.4

0.221 5.6 y 4.0 0.6 1.4 l

1,186 58.1 I 57.3 0.3 3.0 liver 0.518 21.6 13.8 7.2 1.6 cancer 0.197 4.3 1.4 0.3 2.3 1,547 79.8! 82.1 1.1 1.2 0.374 13.9 y 11.8 0.3 2.6 0.473 19.2 20.5 0.5 1.5

1,368 68.8 73.0 2.3 1.2 I.

__i \

0.175 3.1 1.6 0.3 1.6 I.

0.582 25.0 4.9 0.6 20.3 0.207 4.8 2.7 1.4 1.2 0.313 10.6 3.3 7.2 1.8 stomach 0.481 19.6 5.2 0, 3 ^ 5.9 cancer 0.475 19.3 17.1 0.7 2.2 0.238 6.5 1.1 2.4 1.3 0.207 4.8 2.3 1.1 1.6 0.322 11.1 4 , 9 5.2 1.5: 0.339 12.0 3.5 0.2 8.1 8302708 -28- TABLE vervolg (continued)

Invention Comparison

Disease Absorption CEA environment

at AFP put CEA HCG

• worth 492 nm | ng / αη ng / am miu / cm ng / cm • 0.582 25.0 13.0 12.2 1.4 y 0.245 6.9 4.5 0.6 1.6 y 0.864 40.2 8.0 32, 2 3.0 y 0.785 35.9 1.6 35.0 1.1 | thick- "0.516 21.5 4.6 17.7 1.2! colon cancer "0.249 7.1 1.2 4.0 2.0 0.192 4.0 1.5 1.3 1.7 0.269 8.2 7.0 0.4 1.1 0 y 239 12.0 μ 2, 6 5.9 4.2 53.8 y '3.3 54.1 1.6 »» * i * T "* 1" ï' [0.232 6.2 | 5.2 0.2 1.0 i 0.265 8.0 7.3 0.4 1.1 ί 0.230 6.1 2.3 0.8 3.0; 0.2 «7.1! 3.4 1.4 2.5 $ benign i 0.177 3, 2 1.7 0.3 1.9 diseases 0.247 7.0 1.9 4.0 1.2 0.484 19.8 4.2 12.4 3.8 0.245 6.9 2.2 0.7 4.0 0.216 5.3 3.8 1.4 1.1 0.203 4.6 2.8 1.8 1.2 8302708

Invention Comparison -29- ΤΑΒΕΧ Η (continued)

Disease Absorption CEA-converted AFP CEA 3 HCG 3 492 nm value ng / cmJ ng / cm miu / cni ng / cm ^ 0/155 2.0 1.2 0.2 1.0 0.165 2.5 2, 0 0.2 1.0 0.168 2.7 1.8 0.3 1.2

Healthy ° '157 X'4 °' 5 ° '7 0.161 2.3 1.0 0.3 1.0 people 0.192 4.0 2.1 0.2 2.0 0.181 3.4 1.0 1.6 1.3 0.155 2.0 0.8 0.3 1.0 * 0.157 2.1 0.8 0.4 1.4 0.155 2.0 0.6 0.3 1.4

Table I indicates the percentages of positives in the patients of each disease, obtained with the individual measurements, assuming that the diagnostic standard values for an expected occurrence of cancer are 10 ng / cm3 or higher for AFP, 5 ng / cm3 or higher for CEA and 3 5 miu / cm or higher for HCG. Table I also includes the percentages positive in the patients of each disease in the measurements in Table H, where, when measuring the total amount of these three substances by the method of the invention}, an absorbance at 492 nm of 0.350 or greater or a CEA conversion value of 8.5 ng / cm3 or higher 10 is considered cancer positive.

8302708 -30-

TABLE I

One or more

Disease Invention AFP CEA HCG, Γ ZÏ * markers 80% 80% 20% 0% 80% liver cancer gastric cancer 60 10 20 30 60 60 10 60 0 60 colon cancer benign 1Q 0 10 0 10 diseases healthy 0 0 0 0 0 people

Furthermore, when the percentages of positives in the patients of each disease were calculated assuming that a patient is cancer positive if at least one of three tumor labels, individually measured, exceeded the default values, * these percentages of positive 5 are also included in Table I; these were found to be the same as the percentages of cancer positive in the case when the three tumor labels were measured as a total. This shows that in a diagnosis of cancer it is not necessary to measure the tumor labels separately, but in practice a diagnosis is possible from the measured value about the total amount.

Example XXI

Measurement of the total amount of fibrinogen and SP ,, a) Production of antibodies to Sepharose 4B as reagent

Anti-fibrinogen antibodies (DAKO Co.) and anti-SP 2 antibodies (DAKO Co.) were diluted with 0.1 M sodium bicarbonate buffer with pH 8.3, respectively, to concentrations of 5 and 3 mg / cm 2, respectively.

3

5 ml portions of CNBr-activated Sepharose 4B (Pharmacia Co.) were added to 5 cm portions of the respective dilutions and each reaction was carried out at room temperature for 2 hours. At the end of the reaction, the reaction mixture was washed with 0.1 M acetate buffer containing 0.5 M sodium chloride and then with PBS, whereby anti-fibrinogen antibodies on sepharose 4B and anti-SP1 antibodies on sepharose 4B were obtained. These were mixed in a 1: 1 ratio to a reagent consisting of various antibodies to Sepharose 4B.

8302708 -31- b) Preparation of standard fibrinogen solutions

Fibrinogen from Sigma Co. was diluted with PBS containing 1% BSA to standard solutions of 80, 40, 20, 10, 5 and 0, respectively.

C) Preparation of SP 2 standard solutions

According to the method of Hans Bohn et al. (Bint 'part 33: 377-378, 1976) 5 kg placenta was extracted with physiological saline and fractionated with Rivanol and ammonium sulfate and purified by an affinity chromatography with 50 cm anti-SP antibodies on Sepharose 4B (1 mg antibody / cm Sepharose) to 3.7 mg purified SP4. This purified SP 2 was diluted with standard PBS containing 1% BSA to standard solutions of 80, 40, 20, 10, 5 and 0 µg / ca., Respectively.

d) Production of enzyme-labeled anti-fibrinogen antibodies and enzyme-labeled anti-SP1 antibodies

The enzyme-labeled anti-fibrinogen antibodies and enzyme-labeled anti-SP1 antibodies were produced from anti-fibrinogen antibody and anti-SP1 antibody according to the procedures of Example Vc ii.

20 e) Measurement of fibrinogen and SP

* 3

0.1 cm portions of fibrinogen and SP, standard solutions at the respective concentrations of 3 according to b) and cl were added to separate test tubes, followed by addition of 0.4 cm portions of a PBS solution containing 1% BSA and 0 2 ml portions of the reagent consisting of antibody-containing sepharose 4B obtained according to a) in this order, after which the reaction was carried out at room temperature for 1 hour each time. At the conclusion of the reaction, the contents of each test tube were centrifuged and washed with distilled water. Separately, enzyme-labeled anti-fibrinogenic antibodies and enzyme-labeled anti-SP1 antibodies of d) were mixed and diluted to a 1500-fold dilution of the enzyme-labeled anti-fibrinogen antibodies and 1000-fold dilution of the enzyme-labeled anti-SP ^ antibodies. 0.5 ml portions of the mixture were added to the test tubes and the mixture was reacted for 1 hour at room temperature in each test tube. After the reaction, the contents of the test tubes were centrifuged and washed with 8302708-32 distilled water, after which 0.5 ml portions of a substrate solution (6 mM / 1 hydrogen peroxide and 20 mM / 1 o-phenylenediamine in PBS ) were added and each mixture reacted for 30 minutes under light exclusion. Then, 2 cm 1N hydrochloric acid was added to each test tube to stop the reaction and the absorbance of the resulting solution measured at a wavelength of 492 nm. The results are listed in Fig. 9.

Example XIII

Measurement of the total amount of AFP, CEA and HCG

A) Preparation of the standard solutions AFP prepared according to example Va, CEA, prepared according to example

Vla and HCG were diluted with PBS containing 1% BSA to solutions 3 3 3 of 80 ng / cm, 80 ng / cm and 80 miu / cm, respectively, and these solutions were mixed in the proportions according to Table K to five different mixed solutions.

TABEE K

Mixing solution AFP CEP HCG

1 111 2 5 11 20 3 1 5 1 4 115 5 3 2. 1

Each of these five mixing solutions was diluted 2-fold, 4-fold and 8-fold with PBS containing 1% BSA to standard solutions.

B) Measurement of AFP, CEA and HCG

Using 0.1 ml portions of the standard solutions obtained according to a) and the mixtures of three different labeled antibodies, the measurement was carried out according to example VIIIc. The respective standard curves are shown in Figure 10.

Even when the standard solutions with AFP, CEA and HCG were used in a mixture, standard curves according to Fig. 1 were obtained. This shows that even if AFP, CEA and HCG are present in a mixture, they can be measured separately and thus the measured value as total amount can be obtained according to the method of the invention.

8302708

Claims (47)

1. Immunological measurement method whereby a sample containing two or more substances to be measured is reacted simultaneously with two or more insolubilized antibodies or antigens and these substances are measured on the basis of an immunological reaction between these substances 5 and the two or more insolubilized antibodies or antigens. Measuring method according to claim 1, characterized in that two or more insolubilized antibodies or antigens are bound together to an insoluble support. Measuring method according to claim 1, characterized in that two or 10 more insoluble antibodies or antigens are bound to separate insoluble carriers. Measuring method according to claim 2, characterized in that the insoluble support is red blood cells, a polymer latex, carbon black or the inner surface of a reaction vessel. Measuring method according to claim 3, characterized in that the insoluble carriers are red blood cells, a polymer latex and carbon black. Measuring method according to claim 1, characterized in that the immunological response is an agglutination reaction or an agglutination inhibition reaction. Measuring method according to claim 2, characterized in that the immunological response is an agglutination reaction or an agglutination inhibition reaction. Measuring method according to claim 3, characterized in that the immunologic reaction is an agglutination reaction or an agglutination inhibition reaction. Measuring method according to claim 1, characterized in that the immunological measuring method is an enzyme immunoassay, a radioimmunoassay or a fluorescence immunoassay. Measuring method according to claim 2, characterized in that the immunological measuring method is an enzyme immunoassay, a radioimmunoassay or a fluorescence immunoassay. 8302708 r -34- Measuring method according to claim 3, characterized in that the immunological measuring method is an enzyme immunoassay, a radioimmunoassay or a fluorescence immunoassay. 12. Measuring method according to claim 9, characterized in that the immunological measuring method is formed by a sandwioh method, a competitive reaction method or an immunometric method. Measuring method according to claim 10, characterized in that the immunological measuring method consists of a sandwich method, a competitive reaction method and an immunometric method. Measuring method according to claim 11, characterized in that the immunological measuring method is a sandwich method, a competitive reaction method or an immunometric method. Measuring method according to claim 1, characterized in that the antibodies are antibodies against tumor markers. Measuring method according to claim 2, characterized in that the antibodies are antibodies against tumor markers. Measuring method according to claim 3, characterized in that the antibodies are antibodies against tumor markers. Measuring method according to claim 6, characterized in that the antibodies are antibodies against tumor markers. Measuring method according to claim 9, characterized in that the antibodies are antibodies against tumor markers. Measuring method according to claim 12, characterized in that the antibodies are antibodies against tumor labels. Measuring method according to claim 1, characterized in that the antibodies are monoclonal antibodies. Measuring method according to claim 2, characterized in that the antibodies are monoclonal antibodies. Measuring method according to claim 3, characterized in that the antibodies are monoclonal antibodies. Measurement method according to claim 6, characterized in that the antibodies are monoclonal antibodies. Measuring method according to claim 9, characterized in that the antibodies are monoclonal antibodies. Measuring method according to claim 12, characterized in that the antibodies are monoclonal antibodies. 8302 7 Ü8 -35- Measuring method according to claim 15, characterized in that the antibodies are monoclonal antibodies. 28. An immunological measuring reagent suitable for reaction with a sample containing two or more substances to be measured, which reagent comprises an S mixture of two or more insolubilized antibodies or antigens. Measuring reagent according to claim 28, characterized in that the two or more insolubilized antibodies or antigens are bound together to one insoluble carrier. Measuring reagent according to claim 28, characterized in that the two or more insolubilized antibodies or antigens are bound to separate insoluble carriers. 31. Measuring reagent according to claim 29, characterized in that the insoluble carrier is constituted by high molecular weight red blood cell latex, carbon black or an internal surface of a reaction container. Measuring reagent according to claim 30, characterized in that the insoluble carrier is red blood cells, a polymer latex or carbon black. Measuring reagent according to claim 28, characterized in that the reagent is suitable for an agglutination reaction or an agglutination inhibition reaction. 34. Measuring reagent according to claim 29, characterized in that the reagent is suitable for an agglutination reaction or an agglutination inhibition reaction. % Measuring reagent according to claim 30, characterized in that the reagent is suitable for an agglutination reaction or an agglutination inhibition reaction. 36. Measuring reagent according to claim 28, characterized in that the reagent is suitable for use in an immunological measuring method in the form of an enzyme immunoassay, a radioimmunoassay or a fluorescence immunoassay. 37. Measuring reagent according to claim 29, characterized in that this reagent is suitable for use in an enzyme immunoassay, a radioimmunoassay or a fluorescence immunoassay. 8302708 r -36- Ί Measuring reagent according to claim 30, characterized in that the reagent is suitable for use in an enzyme immunoassay, a radioimmunoassay or a fluorescence immunoassay. 39. Measuring reagent according to claim 36, characterized in that it is suitable for use in a measuring method according to the sandwich method, a competitive reaction method or an immunometric method. 40. Reagent according to claim 37, characterized in that it is suitable for a sandwich method, a competitive reaction method or an immunometric method. Measuring reagent according to claim 38, characterized in that it is suitable for a sandwich method, a competitive reaction method or an immunometric method. Measurement reagent according to claim 28, characterized in that its antibodies are antibodies against tumor markers. Measuring reagent according to claim 29, characterized in that its antibodies are antibodies against tumor markers. Measuring reagent according to claim 30, characterized in that its antibodies are antibodies against tumor markers. Measuring reagent according to claim 28, characterized in that its antibodies are monoclonal antibodies. Measuring reagent according to claim 29, characterized in that its antibodies are monoclonal antibodies. Measuring reagent according to claim 30, characterized in that its antibodies are monoclonal antibodies. 8302708