GB2125547A - Simultaneous immunoassay of two or more substances - Google Patents

Abstract

An immunological measuring method which comprises reacting a specimen containing two or more substances to be measured with two or more insolubilized antibodies or antigens simultaneously, and measuring the substances based on the immunological reaction brought about between said substances and immobilised reagents.

Description

SPECIFICATION Immunological measuring method and reagent BACKGROUND OF THE INVENTION FIELD OF THE INVENTION This invention relates to a method for simultaneously measuring the total amount of two or more different substances present in one specimen, and a reagent therefor.

DESCRIPTION OF THE PRIOR ART By virtue of the recent progress of clinical medicine, accurate diagnosis and treatment for various diseases has become possible by measuring a number of clinical test items and judging the results comprehensively. However, in the present immunochemical measurements, it takes about 1-4 days for a test on a single item, and hence a great number of days are needed for testing a number of items, and therefore some of the necessary test items have to be omitted from a fear that the chance for necessary prompt medical treatment of the patient might be lost.

Under such circumstances, there is a possibility that the treatment of the patient is not always appropriate.

For instance, as regards cancer which has lately been ranked high in the causes of death, it is believed that measurements of various tumor markers are useful for early diagnosis, comprehension of the condition of the disease judgement of the therapeutic effect, judgement of whether the patient had a return of the disease, etc. In such a case, it is more effective and accurate to conduct measurements of a number of tumor markers and comprehensively judge from the results of the measurements than to judge from the results of the measurement of a single tumor marker. However, as described hereinabove, it takes many days to measure a number of tumor markers. Further, the necessary amount of the specimen (mainly serum, plasma, etc.) is inevitably increased, which increases the patient's pain.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for measuring at one time the total amount of two or more substances present in one speciment by a single measuring operation.

Another object of the present invention is to provide a reagent for measuring at one time the total amount of two or more substances present in one specimen by a single measuring operation.

Accordingly, this invention relates to an immunological measuring method and measuring reagent for measuring at one time the total amount of two or more substances to be measured which comprises reacting a specimen containing substances to be measured with an insolubilized antibody or antigen comprising two or more different antibodies or antigens, and measuring the substances to be measured based on the immunological reaction brought about between them.

In accordance with the present invention, since two or more substances to be measured can be measured at one time, the time required for the measurement can be remarkably reduced and also the amount of the specimen required can be reduced as compared with the conventional methods which involve individual measurements of a number of substances to be measured.

BRIEF DESCRIPTION OF THE DRAWINGS Figure Figure 4 are graphs showing the standard curves in Example 6; Figure 5- Figure 8 are graphs showing the standard curves in Example 10; Figure 9 is a graph showing the standard curves in Example 12; and Figure 10 is a graph showing the dilution curves in Example 1 3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present inventors have discovered that for the early diagnosis of cancer by the measurement of tumor markers, although it is most preferred to conduct individual measurements to find out what kinds of tumor markers are present and in what amounts, it is practically possible to diagnose based on the total amount of the tumor markers as will be made clear in Examples 2, 4, 6 and 11 described hereinbelow. Based on this discovery, the present inventors have continued intensive study in pursuit of a method which enables the quantity of two or more substances present in one specimen to be measured simultaneously as a total amount by a single measuring operation.As a result, they have discovered that the total amount of two or more substances to be measured can be measured by a single measuring operation based on an immunological measuring principle, which measurement is effected by binding two or more different antibodies, corresponding to two or more antigens to be measure, to the same insoluble carrier, or by mixing and using two or more insolublized antibodies obtained by binding antibodies, corresponding to two or more different antigens to be measured, to separate insoluble carriers, without causing any change in the reactivity between each antigen to be measured and the corresponding antibodies. Thus the present invention has been accomplished.

The immunological measuring methods which are the basis for the method of measurement according to this invention are widely employed as methods for measuring physiologically active substances present in extremely small quantities in specimens such as serum, urine, etc. for example, the concentrations of peptide hormones, steroids, proteins, etc., the concentrations of drugs administered to the human body, etc. Among those, the hemagglutinatio reaction and the latex agglutination reaction are favorably employed owing to such advantages as simple measuring operations and shorter time required for the measurement, etc. and enzymeimmunoassay, radioimmunoassay and fluoroimmunoassay are also favorably employed owing to such advantages as high sensitivity, excellent quantitativeness, etc.

The principles of these measuring methods are now briefly described below: (1) Agglutination reaction: When an unknown amount of an antigen to be measured is reacted with a corresponding antibody bound to a particulate carrier(hereinafter referred to as a solid phase) such as red cells, polymer latex, etc., the antigen then binds to the antibody on the solid phase in proportion to its amount present, thereby the solid phase agglutinates. The degree of said agglutination is measured, and the amount of the antigen to be measured is determined by comparing with the degrees of agglutination obtained when measuring the same substance at known concentrations by similar operations.

(2) Sandwich method: When an unknown amount of an unlabelled antigen (antigen to be measured) is reacted with a corresponding antibody bound to a solid phase (first reaction), the antigen to be measured binds to the antibody to form an antigen-antibody complex. When a given amount of a same antibody labelled with a labelling agent is reacted with said antigen-antibody complex (second reaction), the labelled antibody is bound to the aforesaid complex, but a portion of the labelled antibody in excess of the binding capacity of the complex does not bind and remains in the free form.Thereafter, the solid phase is separated from the liquid phase, the activity of the labelling agent in the solid phase or in the liquid phase is measured, and the amount of the unlabelled antigen to be measured is determined based on the standard curve prepared by similar operations using the unlabeiled antigen at known concentrations.

(3) Competitive reaction method: When an unknown amount of an unlabelled antigen (antigen to be measured) and a given amount of a labelled antigen are competitively reacted with a corresponding antibody bound to a solid phase, the unlabelled antigen and the labelled antigen respectively bind to the antibody in proportion to their existing amounts, so that the amount of the labelled antigen which will bind to the antibody will be inversely proportional to the amount of the unlabelled antigen. Thereafter, the solid phase is separated from the liquid phase, and the activity of the labelling agent either in the solid phase or in the liquid phase is measured, and the amount of the antigen is determined based on the standard curve prepared by similar operations using the unlabelled substance at known concentrations.

(4) Immunometric method: When an unknown amount of an unlabelled antigen (antigen to be measured) is reacted with a given amount of a corresponding labelled antibody, and thereafter, the same antigen bound to a solid phase is added to react with the unreacted labelled antibody, the labelled antibody will bind to the antigen on the solid phase in an amount inversely proportional to the amount of the unlabelled antigen. Thereafter, the solid phase is separated from the liquid phase, and the activity of the labelling agent either in the solid phase or in the liquid phase is measured, and the amount of the unlabelled antigen is determined based on the standard curve prepared by similar operations using the unlabelled substance at known concentrations.

This invention provided a method and a reagent for measuring at one time the total amount of two or more substances to be measured present in one specimen by utilizing the abovedescribed principles of the immunological measuring methods.

The measuring method of this invention may be applied to any of the conventional immunological measuring methods which involve binding an antibody or antigen to an insoluble carrier, for example, the hemagglutination reaction, the agglutination inhibition reaction, the competitive reaction method, the sandwich method, the immunometric method, etc. The method of this invention is now schematically described by focusing the agglutination reaction and the sandwich method by way of example.

(1) Agglutination reaction: Where a specimen expected to include four substances A, B, C and D is to 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, are bound to the same insoluble particulate carrier, or they are separately bound to individual insoluble particulate carriers to prepare a suspension or four different suspensions of insoluble particulate carrier carrying said four antibodies. When the specimen is reacted with these suspensions, the substances to be measured A, B, C and D then respectively bind to the corresponding antibodies nn thp. rsrrierR whorrhv thp Annlutinatinn of the narticulate carriers occurs.Bv measurina the degree of this agglutination, the total amount of the two or more substances to be measured present in one speciment may be measured.

(2) Sandwich method: Where a specimen expected to include four substances A, B, C and D is to 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, are bound to the same insoluble carrier, or they are separately bound to individual insoluble carriers to prepare an insoluble carrier or four different insoluble carriers containing said four antibodies. When the specimen is reacted with these insoluble carriers, the respective substances to be measured A, B, C and D respectively bind to the corresponding antibodies on the carriers.After separation of the solid phase, if necessary, labelled antibodies obtained by binding a labelling agent to the respective anti-A antibody, anti-B antibody, anti-C antibody and anti-D antibody are reacted with the solid phase, thereby the respective labelled antibodies bind to the corresponding substances to be measured respectively. Thereafter, the solid phase is separated, and then the total activity of the labelling agent bound to the solid phase is measured. Thus, the total amount of the two or more substances to be measured present in one specimen may be measured at one time.

The insoluble particular carrier to be used in the agglutination method of this invention may be any of those conventionally employed. That is, cells of e.g. bacteria, red blood cells etc., organic polymer latexes such as polystyrene latex, carboxyl group-incorporated polystyrene latex, styrene-divinylbenzene copolymer latex, hydroxyl group or carboxyl group-incorporated styrenedivinylbenzene polymer latex, polyvinyl alcohol latex, polyacrylate latex, vinyl acetate-acryl copolymer latex, etc., inorganic substances such as silica, carbon black, alumina etc., may be employed either alone or in combination.

As the material for the insoluble carrier in the sandwich method, the competitive reaction method and the immunometric method according to this invention, any of those employed in the conventional immunological measurements, for example, polystyrene, polyethylene, polyacryl, teflon, paper, glass, agarose, etc., may be employed either alone or in combination.

Further, the shape to be employed may suitably be, for example, a sphere, rod, disc, or container form, e.g. an optical cell, test tube, etc., although other shapes can also be employed.

Antibodies used in the measuring method of this invention can be ordinary multi-clonal antibodies, but the use of monoclonal antibodies gives even better results. For example, monoclonal antibodies result in improvement of the sensitivity and accuracy of the measurement, reduction in the reaction time, improvement of the specificity, simplification of the measuring operations, exclusion of any non-specific reactions, exclusion of any inhibitions on the reaction due to the body components in the specimen, e.g. serum, urine etc., and the like.

When the monoclonal antibodies are to be employed in the agglutination reaction, it is necessary to use in combination two or more monoclonal antibodies corresponding to different antigen sites of one substance to be measured in order to cause the agglutination.

As the method for binding one or more antibodies or antigens to an insoluble carrier, the methods described in Clinica Chimica Acta, 48:15 (1973); Journal of Immunology, 116:1554 (1976); and Science, 158:1570 (1967) may be employed. Where two or more different antibodies are to be bound to a single insoluble carrier, an antibody solution obtained by mixing the antibodies to be bound in an appropriate proportion, for example, a solution containing 0.5 mg/ml of anti-AFP antibody, 0.1 mg/ml of anti-HCG antibody and 0.25 mg/ml of anti-CEA antibody in 0.05 M phosphate buffered physiological saline, pH 6.4, is prepared beforehand, then this antibody mixed solution is brought into contact with the insoluble carriers and the reaction is effected at 30-56"C for 2-3 hours.After the reaction, the carriers were washed with physiological saline to prepare antibody-bound carriers.

Where anti-AFP antibody, anti-HCG antibody and anti-CEA antibody are to be individually bound to separate insoluble carriers, said anti-AFP antibody, anti-HCG antibody and anti-CEA antibody are individually dissolved in 0.05 M phosphate buffered physiological saline, pH 6.4 at concentrations of 0.5 mg/ml, 0.1 mg/ml and 0.25 mg/ml respectively, then brought into contact with separate insoluble carriers, and each mixture is reacted at 30-56'C for 2-3 hours.

After the reaction insoluble carriers are washed with physiological saline to prepare respective antibody-bound carriers. The mixing ratio of the thus obtained insoluble antibodies is suitably chosen from the range of about 1-10:1-10:1-10 although it varies depending on the amount of each antibody to be bound to the insoluble carrier and the titer of each antibody.

The amount of each antibody to be bound to the insoluble carrier is suitably 10-0.05 mg/ml, preferably 5-0.1 mg/ml, although it varies depending on the kind of each substance to be measured, the amount of each substance to be measured present in the specimen, the titer of each antibody, etc.

When the measurement is conducted, the optimum conditions are experimentally determined since the amount of the specimen to be used, the amount of each labelled antibody or labelled antigen, and the conditions such as the reaction time, the temperature, etc., vary depending on the nature of each substance to be measured, the titer of each antibody used, and the nature of the labelling agent etc.

The production of the labelled antibody or labelled antigen may be by the conventional process.

Where the method of this invention is to be conducted based on the competitive reaction, the labelled antigen to be used is that which will bind with the insolubilized antibody competitively with the substance to be measured, so that in general, the same substance es the s;bs^nvd be measured is employed as the antigen. Hov > evsr, the physiologically acid subst-ncss, present in the human body, might exist bound to other body components, or unde; > e partial metabolism and thus are sometimes slightly different from the case where they are present out of the human body.Therefore, in such a case, a substance which has substantially the slime reactivity from an immunological viewpoint may also be employed as the identical substance to be measured.

Although the present invention aims simultaneously to measure two or more substances, it is also possible to measure a single substance by using only one labelled antibody, even when two or more insolubilized antibodies are employed unless the substances to be measured interfere with each other.

As the labelling agent, enzymes (e.g. peroxidase, ssgalactosidase, alkaline phcsphates, glucose oxidase etc.), radioactive isotopes (e.g. 1251, 3H, etc.), fluorescent substances (e.g.

fluorescein isothiocyanate, tetramethyl rhodamine isothiocyanate, etc.), etc. may be employed.

In the agglutination reaction, by employing the carrier carrying two or more antibodies obtained as described hereinabove, the total amount of the two or more substances to be measured may be measured at one time. In the sandwich method and the competitive reaction method, by employing two or more labelled antigens or labelled antibodies corresponding to two or more antibodies bound to the insolubilized carriers as described hereinabove, the total amount of the two or more substances to be measured may be measured at one time. Further, in the sandwich method and the competitive reaction method, by emp!oying a single labelled antigen or labelled antibody, the sole measurement of one substance is also possible. Fcr example, a specimen diluted to an appropriate concentration is added to a test tube where an antibody has been bound, and the antigen-antibody reaction is effected.After completion of the reaction, the test tube is washed with distilled water, a labelled antibody is added and the reaction is effected. Then, after washing with distilled water, the activity of the labelling agent in the solid phase (test tube) is measured by a suitable means. From the measured value obtained, the amount of the substances to be measured present in the specimen is calculated based on the standard curve obtained by similar operations on the standard substance of the known concentrations. The standard substance in this case may be either a single antigen or a mixture of a plurality of antigens.

The measuring method of this invention can measure any substance which could be measured by the conventional immunological measuring methods. Especially important substances to be measured are, for example, tumor markers which have an important significance in the early diagnosis of tumors, the judgement of the therapeutic effect thereon, etc.

Examples thereof include carcinoembryonic antigen (hereinafter referred to as CEA), a fetoprotein (AFP), human chorionic gonadotropin (HCG), ss2-microglobulin (fi2m), basic fetopro- tein (BFP), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (γ;-GTP), fregnacy associated ss2-glycoprotein (SP1), pregnancy-associated a2-glycoprotein (SP3), immunosuppressive acidic protein (IAP), immunosuppressive a2-macrogiobulin, acidic isoferritin, fib,inogen, haptogobin, calcitonin, steroid hormones, polyamines, DNA-binding proteins, a1-antitrypsin, pancreatic oncofetal antigen, galactosyl transferase (GT-II), etc.; as well as many substances presently under study.Furthermore, as substances having a significance in the diagnosis and prophyl3xis of hepatitis caused by the infection with hepatitis virus after blood transfusion, there are hepatitis B virus antigens (HBs, HBc, 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) SP3, fibrinogen, (3) AFP, CEA, HCG, SP3, fibrinogen, (4) fibrinogen, haptoglobin, ferritin, (5) haptoglobin, ss2m, immunosuppressive a2-macroglobulin, a,-antitrypsin, (6) ALP, y-GTP, GT-II, (7) polyamines, steroid hormones, (8) HBs, HBc, HBe, and the like.

The accomplishment of the present invention enables the measuring time to be shortened, otherwise, when a number of antigens were to be measured by a conventional method, individual antigens have to be separately measured and therefore a prolonged time was needed.

Furthermore, the amount of the specimen required nas now been yreatly reduced.

This invention is now more particularly described by the following examples.

Example 1 Measurement of Total Amount of Fibrinogen, Haptoglobin and Ferritin a) Production of Antibody-bound Latex Reagent Anti-fibrinogen antibody (DAKO Co.), anti-haptoglobin antibody (DAKO Co.) and anti-ferritin antibody (DAKO Co.) were diluted and mixed r. t 591 phosphate buffered phys, olo3tcai irs, pH 6.4 (hereinafter referred to as PBS) so as to give concentrations of 5 mg/ml, 1.8 mg/m.

and 0.7 mg/ml, respectively. 0.5 ml of 10% polystyrene latex suspension (uniform latex particles, Dow Chemical Co.) was added to 2 ml of the above mixed antibody solution, and stirred and reacted at 37"C for 2 hours. After completion of the reaction, the reaction mixture was cooled with ice, centrifuged, washed with PBS, and suspended in PBS containing 1% bovine serum albumin (hereinafter referred to as BSA), to produce an antibody-bound latex reagent.

b) Preparation of Standard Solutions Fibrinogen (Sigma Co.), haptoglobin (Sigma Co.) and ferritin (Sigma Co.) were individually diluted with PBS containing 1% BSA to concentrations of 120, 60, 30, 1 5 and 0 yg/ml, 12, 6, 3, 1.5 and 0 mg/ml, and 0.8, 0.4, 0.2, 0.1 and 0,ug/ml, respectively.

c) Measurement of Fibrinogen, Haptoglobin and Ferritin 20 yl of each of the solutions of fibrinogen, haptoglobin and ferritin of the respective concentrations produced in b) above was placed on a glass slide, then 50 ,ul of PBS containing 1% BSA and 20 ,tel of the antibody-bound latex reagent produced in a) above were added. The mixture was reacted with stirring for 3 minutes, and the degree of agglutination was observed microscopically. The reaction showing no agglutination was designated negative (-), and that showing agglutination was designated positive (+, + + or + + + according to the degree) thus giving four ranks. The sensitivity of the reagent to fibrinogen, haptoglobin and ferritin was 15 pg/ml, 1.5 mg/ml and 0.1 yg/ml respectively.

Example 2 Measurements on serum specimens from patients Using serum specimens obtained from 1 8 liver cancer patients, 20 stomach cancer patients, 20 large intestine cancer patients, 8 lung cancer patients, 25 various benign disease patients and 20 healthy humans, the measurements according to this invention were conducted. The procedures of the measurements were similar to those in Example 1 c). The results are given in Table 1.

Table 1 No. of Judgement Positive ratio Name of Disease Patients - + + + + + + (%) Liver Cancer 18 1 2 9 6 94 Stomach Cancer 20 3 4 8 5 85 Large Intestine Cancer 20 2 3 7 8 90 Lung Cancer 8 0 2 4 2 100 Benign Diseases 25 19 5 1 0 24 Healthy Humans 20 18 2 0 0 10 As seen in Table 1, cancer patients showed a high postive ratio which indicates the usefulness of the measuring method of the present invention in screening cancer patients.

Example 3 Measurement of Total Amount of Fibrinogen, Haptoglobin and Ferritin a) Production of Anti-fibrinogen Antibody-bound Latex Anti-fibrinogen antibody (DAKO Co.) was diluted with PBS to give a concentration of 5 mg/ml. To 2 ml of the diluted antibody was added 0.5 ml of a 10% polystyrene latex suspension (average particle diameter 0.48 , Dow Chemical), and the mixture was stirred and reacted at 37"C for 2 hours. After completion of the reaction, the reaction mixture was cooled with ice, centrifuged, washed with PBS, and then suspended in PBS containing 1% BSA to produce an anti-fibrinogen antibody-bound latex.

b) Production of Anti-haptoglobin Antibody-bound Latex Anti-haptoglobin antibody (DAKO Co.) was diluted with PBS to give a concentration of 1.8 mg/ml, and this solution was used to react with a 10% polystyrene latex suspension similarly as in a) above to produce an anti-haptoglobin antibody-bound latex.

c) Production of Anti-ferritin Antibody-bound Latex Anti-ferritin antibody (DAKO Co.) was diluted with PBS to 1.2 mg/ml, and this solution was reacted with a suspension of 10% polystyrene latex having incorporated carboxyl groups (average particle diameter 0.25 ,Lt, Dow Chemical) similarly as in a) above to produce an antiferritin antibody-bound latex.

d) Production of Antibody-bound Latex Reagent The anti-fibrinogen antibody-bound latex, anti-haptoglobin antibody-bound latex and anti ferritin antibody-bound latex produced respectively in a), b) and c) above were mixed in a ratio of 2:5:1 to produce an antibody-bound latex reagent.

e) Measurement of Fibrinogen, Haptoglobin and Ferritin 20 ,zl of each of the standard solutions of fibrinogen, haptoglobin and ferritin produced in Example 1 b) were placed on a glass slide, then 50 ,ul of PBS containing 1% BSA and 2O /li of the antibody-bound latex reagent produced in d) above were added to the standard solution on the glass slide. This mixture was reacted with stirring for 3 minutes, and the degree of agglutination was observed microscopically. The reaction showing no agglutination was designated negative (-), and that showing agglutinetion was designated positive (+, + + or + + + according to the degree) thus giving four ranks.The sensitivity of the reagent to fibrinogen, haptoglobin and ferritin was 1 5 jug/ml, 1.5 mg/ml and 0.1 yg/ml respectively.

Example 4 Measurements on serum specimens from patients Serum specimens obtained from 1 5 liver cancer patients, 20 stomach cancer patients, 1 7 large intestine cancer patients, 1 2 lung cancer patients, 25 various benign disease patients and 20 healthy humans were measured using the reagent produced in Example 3. The results are given in Table 2.

Table 2 No. of Judgement Positive ratio Name of Disease Patients - + + + + + + (%) Liver Cancer 15 1 2 7 5 93 Stomach Cancer 20 3 4 8 5 85 Large Intestine Cancer 17 2 2 6 7 88 Lung Cancer 12 1 2 6 3 92 Benign Diseases 25 20 4 1 0 20 Healthy Humans 20 19 1 0 0 5 As seen in Table 2, cancer patients showed a high positive ratio which indicates the usefulness of the measuring method of the present invention in screening cancer patients.

Example 5 Production of Purified AFP and Anti-AFP Antibody a) Production of Purified AFP 16.2 9 of crude AFP extract were obtained from 51 of the ascites of liver cancer patients by salting out with ammonium sulfate. The crude extract was purified by affinity chromatography using 50 ml of rabbit anti-AFP antibody-bound oepi.arose 4B (1 mg antibody/ml Sepharose) to obtain 924 mg of purified AFP.

b) Production of Monoclonal Anti-AFP Antibody 50 ,ug of the purified AFP produced in a) above was subcutaneously administered together with Freund's complete adjuvant (FCA) to a female BALB/c mouse. The administrations were repeated four times at one week intervals and 4 days after the final administration, the spleen was isolated, and the spleen cells were collected. The spleen cells were washed with Dulbecco's modified MEM medium (hereinafter referred to as D-MEN), 1 X 108 cells were counted and mixed with 1 X 107 cells of mouse myeloma cells (P3-NSI/1-Ag 4-1). They were subjected to cell fusion in one ml of D-MEN containing 42.5% polyethylene glycol 1 540 and 7.5% dimethylsulfoxide at 37"C for 1 minute.To these cells was added HAT medium (RPMI-1640 medium containing hypoxanthin, aminopterin, thymidine and 10% bovine fetus serum) to 20 ml, then the cell mixture was allotted 0.2 ml each to a 96-well microplate and cultured for 2 weeks, after which the antibody activity in the culture supernatant in the proliferated well was measured.

Then, the cells in the wells showing activity were collected and added to 40 ml of RPMI 1 640 medium containing 10% bovine fetus serum and thymus cells from BALB/C mouse. This cell suspension was allotted to two 96-well microplates, and cultured for a week to obtain 9 clones of anti-AFP antibody producing hybridoma. They were transferred to large scale cultures, and one liter of each obtained culture supernatant was subjected to an affinity chromatography using 50 ml of purified AFP-bound Sepharose 4B (0.5 ml AFP/ml Sepharose) to obtain 4.2-11.6 mg of monoclonal antibodies respectively. The respective antibodies were designated Lot No. 1-9.

c) Identification of Antigen-recognition Sites i) Production of Anti-AFP Antibody-bound Test Tubes One ml portions of PBS containing 0.5 mg of the monoclonal anti-AFP antibody Lot No. 1-9 respectively were added to separate polystyrene test tubes which had been washed with PBS, and each test tube was incubated at 37"C for 3 hours to facilitate binding of the antibody to the inner surface of the test tube. After the reaction, the test tubes were washed with PBS to obtain monoclonal antibody-bound test tubes.

ii) Production of Enzyme-labelled Anti-AFP Antibody Five mg of horseradish peroxidase (Behringer Manhelm Co., Grade I; hereinafter referred to as HRPO) were dissolved in 1.0 ml of 0.3 M sodium bicarbonate buffer, and to this solution was added 0.1 ml of 1 % 1 -fluoro-2,4-dinitrobenzene in ethanol and the mixture was reacted for an hour. Further 1.0 ml of 0.06 M sodium periodate solution was added to the mixture, the reaction was effected for 30 minutes, then 1.0 ml of 0.1 6 M ethylene glycol solution was added, and the reaction was continued for an hour. The reaction mixture was dialyzed against 0.01 M sodium carbonate solution, pH 9.5. To this dialyzate solution were added 5 mg of each of the nine lots of anti-AFP antibodies produced in b) above, and each mixture was reacted at room temperature for 3 hours.Then 5 mg of sodium borohydride was added, and the reaction was further continued overnight. The reaction mixtures were respectively dialyzed against 0.01 M PBS, pH 7.2 to obtain each HRPO-labelled anti-AFP antibody.

iii) Identification of Antigen-recognizing Sites To each of the anti-AFP antibody-bound test tubes produced in i) above were added 0.1 ml of the standard solution diluted with PBS so as to contain 100 ng/ml of the AFP produced in a) above and 0.4 ml of a 100-fold dilution of each HRPO-labelled anti-AFP antibody produced in ii) above, and each reaction was effected for 30 minutes. After completion of the reaction, each test tube was washed with a washing solution, 0.5 ml of an enzyme substrate solution containing 20 mg/dl of O-phenylenediamine and 6 mM of hydrogen peroxide was added, and the reaction was effected for 30 minutes. Two ml of 1 N hydrochloric acid were aded to stop the enzyme reaction, and the absorbancy of the reaction mixture at 492 nm was measured.The results are given in Table 3 by assigning + to the combinations achieving the color reaction and - to the combinations not achieving the color reaction.

Table 3 HRPO-labelled Antibody

1 2 3 4 5 6 7 8 9 1 - - - + - + - + + 8 2 - - - + - + - + + c 3 - - - + - + - + + C Qq+ 4 + + + - + + + ~ ~ 5 5 - - - + - + - + + : 6 + + + + + - + + + 0 7 7 - - - + - + - + + 8 + + + - + + + - 9 + + + - + + + - By the difference in the antigen-recognizing sites, the 9 lots of the monoclonal anti-AFP antibodies were classified into 3 groups, i.e. the first group consisting Lots Nos. 1, 2, 3, 5 and 7, the second group consisting of Lot No. 6 and the third group consisting of Lot No. 4, 8 and 9. These groups were designated anti-AFP antibody (A), (B) and (C), respectively. Among those, (A) will be employed as the insolubilized antibody and (C) will be employed as the labelled antibody in the following examples.

Example 6 Production of Purified CEA and Anti-CEA Antibody a) Production of Purified CEA 40 g of large intestine cancer tissue was minced and, after adding 100 ml of distilled water, homogenized in a homogenizer. To this suspension was added the equal volume of 1.2 M perchloric acid and the mixture was extracted with stirring for 30 minutes. The centrifuged supernatant was dialyzed against distilled water to obtain a crude CEA extract.

This crude extract was concentrated to 10 ml and subjected to gel filtration using Sepharose 4B equilibrated with physiological saline and the first protein fraction was collected. Then, the fraction was again applied to gel filtration using Sephadex G-200 similarly equilibrated and the second protein fraction was collected, which was then concentrated to 2 ml to obtain 1 35 /19 of purified CEA.

b) Production of Monoclonal Anti-CEA Antibody Eight clones of anti-CEA antibody producing hybridoma were obtained from the purified CEA produced in a) above by procedures similar to ïnose in Example 5 b). For immunizing the mice, 30 y9 of the purified CEA was employed for each administration.

1 X 106 hybridoma cells were inoculated intraperitoneally in a female BALB/c mouse which had been intraperitoneally given 0.5 ml of pristane (2, 6, 10, 1 4-tetramethylpentadecane; a product of Wako Pure Chemicals, Co., Ltd.). Two weeks later, the ascites was collected. Each ascites was subjected to chromatography on DEAE cellulose equilibrated with 0.01 M phosphate buffer, pH 7.0, to obtain a monoclonal anti-CEA antibody in the unadsorbed fraction. As a result of the identification test of the antigen-recognizing sites according to Example 5 c), the respective antibodies were classified into 3 groups each consisting of 5 lots, 2 lots and 1 lot, which were designated anti-CEA antibody (A), (B) and (C) respectively.Among those, anti-CEA antibody (A) will be used as the insolubilized antibody and anti-CEA antibody (B) will be used as the labelled antibody in the following examples.

Example 7 Preparation of HCG-ss and Anti-HCG-ss Antibody a) Production of HCG-ss Subunit One gram of HCG (2000 lU/mg) was dissolved in 2 ml of 0.025 M phosphate buffer, pH 5.6 and subjected to chromatography on 3 g of DEAE-Sephadex A-50 equilibrated with the same buffer. The fraction eluted with 0.05 M phosphate buffer, pH 5.6 was collected and dialyzed against distilled water to obtain 308 mg of purified HCG, which was then lyophilized.

300 mg of the purified HCG was dissolved in 10 mi of 10 M urea (pH 4.5) and the reaction was effected at 40"C for an hour, followed by chromatography using 2 g of DEAE-Sephadex 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 NaCI and 8 M was dialyzed against physiological saline to obtain 147 mg of HCG-ss subunit.

b) Production of Anti-HCG-ss Antibody Eleven lots of anti-HCG-ss antibody producing hybridoma were obtained from the HCG-ss subunit produced in a) above by similar procedures as in Example 5 b). On the antibodies obtained from these lots, the identification of the antigen-recognizing sites was conducted according to the procedures in Example 5 c) to classify into 2 groups including 8 lots and 2 lots respectively, and designated anti HCG-ss antibody (A) and (B) respectively. Among these anti HCG-ss antibody (A) will be used as the insolubilized antibody and (B) will be used as the labelled antibody in the following examples.When these antibodies were examined for their cross reaction with luteinizing hormone (LH) using the above combination, the reactivity was found to be less than 1 % when the reactivity for HCG was taken as 100%.

Example 8 Measurement of Total Amount of AFP, CEA and PCTJ a) Production of Antibody-bound Test Tube Reagent To polystyrene test tubes were added 1 ml of solution containing 0.1 mg/ml of monoclonal anti-AFP antibody, 0.5 mg/ml of monoclonal anti-CEA antibody and 0.25 mg/ml of monoclonal anti-HCG antibody produced in Examples 5, 6 and 7 respectively, and each test tube was incubated at 37"C for 3 hours. After completion of the incubation, each test tube was washed with PBS to produce reagents comprising antibody-bound test tubes.

b) Preparation of Standard Solutions The AFP produced in Example 5 a), the CEA produced in Example 6 a) and HCG (HCG Mochida; Mochida Pharmaceutical) were diluted with PBS containing 1 % BSA to prepare solutions of 80, 40, 20, 10, 5 and 0 ng/ml, 80, 40, 20, 10, 5 and 0 ng/ml and 80, 40, 20, 10, 5 and 0 miu/ml, respectively.

c) Measurement AFP, CEA and HCG To the reagents comprising antibody-bound rest tubes produced in a) above were added 0.1 ml portions of the AFP, CEA and HCG standard solutions of the resepctive concentrations, followed by the addition of 0.4 ml portions of PBS containing 1 % BSA, and each reaction was effected at room temperature for 2 hours. After completion of the reaction, the test tubes were washed with distilled water. Separately, the enzyme-labelled antibodies produced in Examples 5, 6 and 7 were diluted and mixed in PBS containing 1 % BSA to give a solution containing 2000fold dilution of enzyme-labelled anti-AFP antibody, 800-fold dilution of the enzyme-labelled anti CEA antibody and 500fold dilution of the enzyme-labelled anti-HCG antibody. 0.5 ml portions of said mixture were added to the above test tubes, and each reaction was effected at room temperature for 2 hours. After completion of the reaction, the test tubes were washed with distilled water, 0.5 ml portions of a substrate solution (6 mM/1 of hydrogen peroxide and 20 mM/1 of o-phenylenediamine in PBS) were added, and each reaction was effected at room temperature for 30 minutes while sealing off the light.Further, 2 ml of 1 N hydrochloric acid were added to stop the reaction, and the absorbency of the reaction mixture was measured at a wavelength of 492 nm. For comparison, measurements were also made in the case where the labelled antibodies were not mixed but reacted separately. The results are sumarized in Fig.

1-Fig. 4. The combinations of the insolubilized antibody and the labelled antibody used in the respective figures are as set forth in Table 4.

Table 4 Insolubilized Antibody Labelled Antibody Anti-AFP antibody, Anti-CEA antibody a Fig. 1 Anti-HCG antibody Anti-AFP antibody, in mixture Anti-CEA antibody s Fig. 2 Anti-HCG antibody Anti-AFP antibody alone in mixture Fig. 3 Anti-CEA antibody alone Fig. 4 Anti-HCG antibody alone From the above results, it can be seen that since AFP, CEA and HCG do not show any cross reaction among them, they can be individually measured by employing the respective labelled antibodies even if these substances are present in mixture, and that even when the labelled antibodies are used in mixture, only the respective corresponding antigens and antibodies react and such reactivity is not influenced by the presence of other substances.

Example 9 Measurements on serum specimens from patients Respective serum specimens taken from 10 liver cancer patients, 10 stomach cancer patients, 10 large intestine cancer patients, 10 various benign diseases patients and 10 heatlthy humans were measured for the total amount of AFP, CEA and HCG. Each measurement was made using 0.1 ml of either the standard solution or the serum to be tested by procedures similar as in Example 8 c). The results are expressed by direct absorbancy value at 492 nm and also by CEAconverted value obtained by applying said absorbancy value tentatively to the CEA standard curve. For comparison, AFP, CEA and HCG were also individually measured on the same specimens. The results are given in Table 5.

Table 5 Present Invention Reference CEA Converted Name of Absorbancy Value AFP CEA HCG Disease 492 nm ng/ml ng/ml ng/ml miu/ml 0.570 20.0 20.1 2.1 2.0 0.392 10.7 10.5 5.8 1.8 1.608 85.2 101.8 1.1 3.1 0.231 4.0 2.3 2.2 2.0 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 1.2 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 Stomach 0.492 19.8 1.1 1.7 21.1 Cancer 0.501 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 0.270 5.1 1.0 2.2 3.7 0.260 4.7 2.0 1.0 4.0 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 Large 0.388 10.1 1.5 11.1 0.8 Intestine 0.470 14.8 1.2 13.0 3.2 Cancer 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 0.3 3.0 0.219 3.8 1.2 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 2.2 6.1 1.1 Diseases 0.333 8.5 2.5 1.8 3.5 0.261 4.9 0.8 2.1 3.3 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 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 0.267 4.8 0.7 1.5 3.8 Healthy 0.259 4.7 0.7 2.8 3.3 Humans 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 6 below shows the percentages of positivity among the patients of each disease obtained in the single item measurements when supposing that the diagnostic standard values for the suspected occurrence of cancer are 10 ng/ml or higher for AFP, 5 ng/ml or higher for CEA and 5 miu/ml or higher for HCG.Table 6 also includes the percentages of positivity among the patients of each disease in the measurements in table 5 above, in which, when measuring the total amount of said three substances according to the method of this invention, the absorbancy at 492 nm of 0.350 or higher or its CEA-converted value of 8.5 ng/ml or higher is regarded cancer positive.

Table 6 One or Present More Name of Disease Invention AFP CEA HCG Markers Liver Cancer 80% 80% 20% 0% 80% Stomach Cancer 60 10 20 30 60 Large Intestine Cancer 60 10 60 0 60 Benign Diseases 10 0 10 0 10 Healthy Humans 0 0 0 0 0 Further, when the percentages of positivity among the patients of each disease are calculated assuming that a patient is cancer positive if at least one of the three tumor markers measured individually exceeded the standard values, these percentages of positivity, which are also included in the right column of Table 6, were the same as the percentages of cancer positivity in the case when the three tumor markers were measured as the total amount (the second column of Table 6).This indicates that on diagnosis of cancer, it is not necessary to measure tumor markers individually but, practically, diagnosis is possible from the measured value taken as the total amount.

Example 10 Measurement of Total Amount of AFP, CEA and HCG a) Production of Antibody-bound Polystyrene Bead Reagent The monoclonal anti-AFP antibody, anti-CEA antibody and anti-HCG antibody produced in Examples 5, 6 and 7 respectively were diluted to concentrations of 1.0, 0.8 and 0.5 mg/ml respectively. Polystyrene beads (diameter 2.5 mm) were dipped in each dilution, and each mixture was reacted at 37"C for 3 hours. After completion of the reaction, the beads were washed with PBS to prepare reagents comprising antibody-bound polystyrene beads, each set of reagent consisting of 3 beads each carrying different antibodies.

b) Measurement of AFP, CEA and HCG To test tubes (inner diameter 10 mm, length 60 mm) were added 0.1 ml- portions of the AFP, CEA and HCG standard solutions of the respective concentrations prepared in Example 8 b), and 0.4 ml portions of PBS solution containing 1 % BSA, and a set of the reagent comprising three different antibody-bound polystyrene beads produced in a) above were added thereto. After stirring, the reaction was effected at room temperature for 2 hours. After completion of the reaction, the polystyrene beads were washed with distilled water.Separately, the enzymelabelled antibodies produced in Examples 5, 6 and 7 were diluted and mixed in PBS containing 1 % BSA to give a solution containing 2500-fold dilution of the enzyme-labelled anti-AFP antibody, 1000-fold dilution of the enzyme-labelled anti-CEA antibody and 650-fold dilution of the enzyme-labelled anti-HCG antibody. 0.5 ml portions of said mixture were added to the above beads, and each reaction was effected at room temperature for 2 hours. After completion of the reaction, the polystyrene beads were washed with distilled water, 0.5 ml portions of a substrate solution (6 mM/l of hydrogen peroxide and 20 mM/I of o-phenylene-diamine in PBS) were added, and each mixture was reacted at room temperature for 30 minutes while sealing off the light. Further, 2 ml of 1 N hydrochloric acid were added to stop the reaction, and the absorbancy was measured at a wavelength of 492 nm. For comparison, measurements were also made in the case where the labelled antibodies were not mixed but reacted separately. The results are summarized in Fig. 5-Fig. 8. The combinations of the insolubilized antibody and the labelled antibody used in the respective figures are as set forth in Table 7.

Table 7

I nsolubilized Antibody Labelled Antibody Anti-AFP antibody, Anti-CEA antibody 8 Fig. 5 Anti-HCG antibody Mixture of three beads in mixture Anti-AFP antibody bound bead Fig. 6 ( Anti-CEA antibody bound bead 8 Anti-AFP antibody aíone Anti-HCG antibody bound bead Fig. 7 Anti-CEA antibody alone Fig. 8 Anti-HCG antibody alone From the above results, similarly as in Example 8, it can be seen that since AFP, CEA and HCG do not show any cross reaction among them, they may be individually measured by employing the respective labelled antibodies even if these substances are present in mixture, and that even when the labelled antibodies are in mixture, only the respective corresponding antigens and antibodies react and such reactivity is not influenced by the presence of other substances.

Example 11 Measurements on serum specimens from patients Respective serum specimens taken from 10 liver cancer patients, 10 stomach cancer patients, 10 large intestine cancer patients, 10 various benign diseases patients and 10 healthy humans were measured for the total amount of AFP, CEA and HCG. Each measurement was made using 0.1 ml of either the standard solution or the serum to be tested by procedures similar as in Example 8 c). The results are expressed by direct absorbancy value at 492 nm and also by CEAconverted value obtained by applying said absorbancy value tentatively to the CEA standard curve. For comparison, AFP, CEA and HCG were also individually measured on the same specimens. The results are given in Table 8.

Table 8 Present Invention Reference CEA Converted Name of Absorbancy Value AFP CEA HCG Disease 492 nm ng/ml ng/ml ng/ml miu/ml 1.55 < 80 < 320.5 9.6 1.2 1.55 < 80 < 173.6 3.0 1.4 0.221 5.6 4.0 0.6 1.4 1.186 58.1 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 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 0.175 3.1 1.6 0.3 1.6 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 15.9 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 0.582 25.0 13.0 12.2 1.4 0.245 6.9 4.5 0.6 1.6 0.864 40.2 8.0 32.2 3.0 Large 0.785 35.9 1.6 35.0 1.1 Intestine 0.516 21.5 4.6 17.7 1.2 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 0.339 12.0 2.6 5.9 4.2 1.110 53.8 3.3 54.1 1.6 0.232 6.2 5.2 0.2 1.0 0.265 8.0 7.3 0.4 1.1 0.230 6.1 2.3 0.8 3.0 0.249 7.1 3.4 1.4 2.5 Benign 0.177 3.2 1.7 0.3 1.9 Disease 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 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 0.157 2.1 1.4 0.5 0.7 Healthy 0.161 2.3 1.0 0.3 1.0 Humans 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 9 below shows the percentages of positivity among the patients of each disease obtained in the single item measurements when supposing that the diagnostic standard values for the suspected occurrence of cancer are 10 ng/ml or higher for AFP, 5 ng/ml or higher for CEA and 5 miu/ml or higher for HCG.Table 9 also includes the percentages of positivity among the patients of each disease in the measurements in Table 8 above, in which, when measuring the total amount of said three substances accor-iing to the method of this invention, the absorbancy at 492 nm of 0.350 or higher or its CEA converted value of 8.5 ng/mi or higher is regarded cancer positive.

Table 9 One or Present More Name of Disease Invention AFP CEA HCG Markers Liver Cancer 80% 80% 20% 0% 80% Stomach Cancer 60 10 20 30 60 Large Intestine Cancer 60 10 60 0 60 Benign Disease 10 0 10 0 10 Healthy Humans 0 0 0 0 0 Further, when the percentages of positivity among the patients of each disease are calculated assuming that a patient is cancer positive if at least one of the three tumor markers measured individually exceeded the standard values, these percentages of positivity, which are also included in table 9, were the same as the percentages of cancer positivity in the case when the three tumor markers were measured as the total amount. This indicates that on diagnosis of cancer, it is not necessary to measure tumor markers individually but, practically, diagnosis is possible from the measured value taken as the total amount.

Example 12 Measurement of Total Amount of Fibrinogen and SP3 a) Production of Antibody-bound Sepharose 4B Reagent Anti-fibrinogen antibody (DAKO Co.) and anti-S P3 antibody (DAKO Co.) were respectively diluted with 0.1 M sodium bicarbonate buffer, pH 8.3, to give concentrations of 5 and 3 mg/ml respectively. To 5 ml portions of the respective dilutions were added 5 ml portions of CNBr-activated Sepharose 4B (Pharmacia Co.), and each reaction was effected at room temperature for 2 hours. After completion of the reaction, the reaction mixture was washed with 0.1 M acetate buffer containing 0.5 M sodium chloride and then with PBS, thereby antifibrinogen antibody-bound Sepharose 4B and anti-SPq antibody-bound Sepharose 4B were prepared.They were mixed in a ratio of 1:1 to produce a reagent comprising antibody-bound Sepharose 4B.

b) Preparation of Fibrinogen Standard Solutions Fibrinogen (Sigma Co.) was diluted with PBS containing 1 % BSA to prepare standard solutions of 80, 40, 20, 10, 5 and 0,ug/ml respectively.

c) Preparation of SP3 Standard Solutions According to the method by Hans Bohn et al. (Blut Band 33:377-378, 1976), 5 kg of placenta was extracted with physiological saline, fractionated using Rivanol and ammonium sulfate, and purified by affinity chromatography using 50 ml of the anti-S P3 antibody-bound Sepharose 4B (1 mg antibody/ml Sepharose) to obtain 3.7 mg of purified SP3. This purified SP3 was diluted with PBS containing 1 % BSA to prepare standard solutions of 80, 40, 20, 10, 5 and 0 yg/ml respectively.

d) Production of Enzyme-labelled Anti-fibrinogen Antibody and Enzyme-labelled Anti-S P3 Antibody.

The enzyme-labelled antifibrinogen antibody and enzyme-labelled anti-S P3 antibody were produced from the anti-fibrinogen antibody and anti-S P3 antibody by similar procedures as in Example 5 c) ii).

e) Measurement of Fibrinogen and SP3 To separate test tubes were added 0.1 ml portions of the fibrinogen and SP3 standard solutions of the respective concentrations produced in b) and c) above, followed by the addition of 0.4 ml portions of PBS solution containing 1% BSA and 0.2 ml portions of the reagent comprising antibody-bound Sepharose 4B produced in a) above successively, and, after stirring, each reaction was effected at room temperature 3 for an hour. After the reaction, the contents in the test tubes were centrifuged and washed with distilled water.Separately, the enzyme-labelled anti-fibrinogen antibody and enzyme-labelled anti-S P3 antibody produced in d) above were mixed and diluted to contain 1500-fold dilution of the enzyme-labelled anti-fibrinogen antibody and 1000-fold dilution of the enzyme-labelled anti-SP3 antibody. 0.5 ml portions of the mixture were added to the above test tubes, and the mixture in each test tube was reacted at room temperature for an hour. After completion of the reaction, the contents of the test tube were centrifuged and washed with distilled water, and, after adding 0.5 ml portions of a substrate solution (6 mM/l of hydrogen peroxide and 20 mM/l of o-phenylenediamine in PBS), each mixture was reacted at room temperature for 30 minutes while sealing off the light.Further, 2 ml of 1 N hydrochloric acid was added to stop the reaction, and the absorbancy of the resulting solution was measured at a wavelength of 492 nm. The results are given in Fig. 9.

Example 13 Measurement of Total Amount of AFP, CEA and HCG a) Preparation of Standard Solutions The AFP produced in Example 5 a), the CEA produced in Example 6 a) and HCG (HCG Mochida) were diluted with PBS containing 1 % BSA to prepare solutions of 80 ng/ml, 80 ng/ml and 80 miu/ml respectively, and these solutions were mixed in the various proportions set forth in Table 10 to prepare 5 different mixed solutions.

Table 10 No. of Mixed solution AFP CEP HCG 1 1 1 1 2 5 1 1 3 1 5 4 1 1 5 5 3 2 1 Each of the above 1-5 mixed solution was diluted 2-fold, 4-fold and 8-fold with PBS containing 1 % BSA to prepare standard solutions.

b) Measurement of AFP, CEA and HCG Using 0.1 ml portions of the standard solutions produced in a) above and the mixtures of three different labelled antibodies, measurement was conducted in a similar manner as in Example 8 c). The respective standard curves are shown in Fig. 10. Even when the standard solutions containing AFP, CEA and HCG in mixture were employed, the standard curves similar to Fig. 1 were obtained. This indicates'that, even when AFP, CEA and HCG are present in mixture, these may be measured individually and hence the measured value as their total amount may be obtained by the method of this invention.

Claims (47)

1. An immunological measuring method comprising reacting a specimen containing two or more substances to be measured with two or more insolubilized antibodies or antigens simultaneously and measuring said substances based on an immunological reaction brought about between said substances and said two or more insolubilized antibodies or antigens.

2. A measuring method according to Claim 1, wherein said two or more insolubilized antibodies or antigens are bound to an insoluble carrier together.

3. A measuring method according to Claim 1, wherein said two or more insolubilized antibodies or antigens are bound to separate insoluble carriers respectively.

4. A measuring method according to Claim 2, wherein the insoluble carrier is selected from the group consisting of red blood cells, polymer latex, carbon black and an inner surface of a reaction vessel.

5. A measuring method according to Claim 3, wherein the insoluble carriers are selected from the group consisting of red blood cells, polymer latex and carbon black.

6. A measuring method according to Claim 1, wherein said immunological reaction is an agglutination reaction or an agglutination inhibition reaction.

7. A measuring method according to Claim 2, wherein said immunological reaction is an agglutination reaction or an agglutination inhibition reaction.

8. A measuring method according to Claim 3, wherein said immunological reaction is an agglutination reaction or an agglutination inhibition reaction.

9. A measuring method according to Claim 1, wherein said immunologicl measuring method is enzymeimmunoassay, radio-immunoassay or fluoroimmunoassay.

10. A measuring method according to Claim 2, wherein said immunological measuring method is enzymeimmunoassay, radio-immunoassay or fluoroimmunoassay.

11. A measuring method according to Claim 3, wherein said immunological measuring method is enzymeim munoassay, radio-immunoassay or fluoroimmunoassay.

1 2. A measuring method according to Claim 9, wherein the immunological measuring method is further selected from the group consisting of a sandwich method, competitive reaction method and immunometric method.

13. A measuring method according to Claim 10, wherein the immunological measuring method is further selected from the group consisting of a sandwich method, competitive reaction method and immunometric method.

1 4. A measuring method according to Caim 11, wherein the immunological measuring method is further selected from the group consisting of a sandwich method, competitive reaction method and immunometric method.

1 5. A measuring method according to Claim 1, wherein said antibodies are antibodies to tumor markers.

1 6. A measuring method according to Claim 2, wherein said antibodies are antibodies to tumor markers.

1 7. A measuring method according to Claim 3, wherein said antibodies are antibodies to tumor markers.

1 8. A measuring method according to Claim 6, wherein said antibodies are antibodies to tumor markers.

1 9. A measuring method according to Claim 9, wherein said antibodies are antibodies to tumor markers.

20. A measuring method according to Claim 12, wherein said antibodies are antibodies to tumor markers.

21. A measuring method according to Claim 1, wherein the antibodies are monoclonal antibodies.

22. A measuring method according to Claim 2, wherein the antibodies are monoclonal antibodies.

23. A measuring method according to Claim 3, wherein the antibodies are monoclonal antibodies.

24. A measuring method according to Claim 6, wherein the antibodies are monoclonal antibodies.

25. A measuring method according to Claim 9, wherein the antibodies are monoclonal antibodies.

26. A measuring method according to Claim 12, wherein the antibodies are monoclonal antibodies.

27. A measuring method according to Claim 15, wherein the antibodies are monoclonal antibodies.

28. An immunological measuring reagent adapted for reacting with a specimen containing two or more substances to be measured by an immunological reaction, said reagent comprising a mixture of two or more insolubilized antibodies or insolubilized antigens.

29. A measuring reagent according to Claim 28, wherein said two or more insolubilized antibodies or insolubilized antigens are bound te an insoluble carrier together.

30. A measuring reagent according to Claim 28, wherein said two or more insolubilized antibodies or insolubilized antigens are bound to separate insoluble carriers respectively.

31. A measuring reagent according to Claim 29, wherein the insoluble carrier is selected from the group consisting of red blood cells, high molecular weight latex, carbon black and an inner surface of a reaction vessel.

32. A measuring reagent according to Claim 30, wherein the insoluble carriers are selected from the group consisting of red blood cells, polymer latex and carbon black.

33. A measuring reagent according to Claim 28, wherein said reagent is adapted for reacting by an agglutination reaction or an agglutination inhibition reaction.

34. A measuring reagent according to Claim 29, wherein said reagent is adapted for reacting by an agglutination reaction or an agglutination inhibition reaction.

35. A measuring reagent according to Claim 30, wherein said reagent is adapted for reacting by an agglutination reaction or an agglutination inhibition reaction.

36. A measuring reagent according to Claim 28, wherein said reagent is adapted for use in an immunological measuring method selected from the group consisting of enzymeimmunoassay, radioimmunoassay and fluoroimmunoassay.

37. A measuring reagent according to Claim 29, wherein said reagent is adapted for use in an immunological measuring method selected from the group consisting of enzymeimmunoassay, radioimmunoassay and fluoroimmunoassay.

38. A measuring reagent according to Claim 30, wherein said reagent is adapted for use in an immunological measuring method selected from the group consisting of enzymeimmunoassay, radioimmunoassay and fluoroimmunoassay.

39. A measuring reagent according to Claim 36, wherein said immunological measuring method is further selected from the group consisting of sandwich method, competitive reaction method and immunometric method.

40. A measuring reagent according to Claim 37, wherein said immunological measuring method is further selected from the group consisting of sandwich method, competitive reaction method and immunometric method.

41. A measuring reagent according to Claim 38, wherein said immunological measuring method is further selected from the group consisting of sandwich method, competitive reaction method and immunometric method.

42. A measuring reagent according to Claim 28, wherein said antibodies are antibodies to tumor markers.

43. A measuring reagent according to Claim 29, wherein said antibodies are antibodies to tumor markers.

44. A measuring reagent according to Claim 30, wherein said antibodies are antibodies to tumor markers.

45. A measuring reagent according to Claim 28, wherein said antibodies are monoclonal antibodies.

46. A measuring reagent according to Claim 29, wherein said antibodies are monoclonal antibodies.

47. A measuring reagent according to Claim 30, wherein said antibodies are monoclonal antibodies.