FR2531223A1 - IMMUNOLOGICAL ASSAY PROCESS AND REAGENT - Google Patents

Abstract

IN AN IMMUNOLOGICAL DETERMINATION METHOD WHICH CONSISTS OF REACTING A SAMPLE CONTAINING ONE OR MORE SUBSTANCES TO BE DOSE WITH AN INSOLUBILIZED ANTIBODY OR ANTIGEN AND DETERMINING THE SUBSTANCE (S) TO BE DETERMINED BASED ON THE IMMUNOLOGICAL REACTION WHICH OCCURS BETWEEN THEM, THE THE PRESENT INVENTION RELATES TO AN IMPROVED IMMUNOLOGICAL ASSESSMENT PROCESS FOR DETERMINING ON ONE TIME THE TOTAL QUANTITY OF TWO OR MORE SUBSTANCES TO BE DOSED, IN WHICH TWO OR MORE ANTIBODIES OR ANTIGENS ARE USED AS AN INSOLUBILIZED OR ANTIGENES ANTIBODIES. THE PRESENT INVENTION ALSO CONCERNS THE REAGENT USED IN THIS ASSAY PROCESS.

Description

1 - The present invention relates to a method for the simultaneous dosing of

  the total amount of two or more different substances present in a sample, and one

reactive for this process.

  Thanks to recent advances in clinical medicine, it has become possible to make an accurate diagnosis,

  as well as the resulting treatment, for various illnesses

  dies, by measuring a number of clinical parameters

  and judging the results - overall Cepen-

  Dant, in current immunochemical assays, it is necessary to

  1 to 4 days approximately to carry out a test on only one pa-

  reduces, and therefore several days are necessary for your-

  ter a number of parameters, and therefore some

  some necessary tests should be omitted when a treatment

  prompt medical attention is required Under these conditions, treatment may not always be the same

  which would best suit the patient.

  For example, in the case of cancer, which has recently ranked as one of the leading causes of death, it is believed that the assays of various markers of

  tumors are useful for early diagnosis, for com-

  grasp of the state of the disease, to judge the therapeutic effect, to recognize a relapse of the disease, etc. In such a case, the determination of a certain number of tumor markers and the overall study of the results of these dosages offer more efficiency and precision than

  the dosage and the study of the results -of the dosage of a 'single market-

  tumor tumor However, as mentioned above, it takes several days to measure several markers of

  tumors In addition, the amount of sample required (main-

  of course serum, plasma, etc.) is inevitably more

  large, which is inconvenient for the patient.

  One of the aims of the present invention is to provide a method for dosing at once the total amount of two or more substances present in a single

  sampled by a single dosing operation.

  Another b-_t of the present invention is to-

  set a reagent nor dose in one go the total amount of two or more substances present in a

  single sample By one dosing operation.

  Accordingly, the present invention relates to an immunoassay method and a reagent for assaying the total amount of two or more substances to be assayed at once, which method consists in reacting a Llon sample containing the substances to be assayed with a insolubilized antibody or antigen consisting of two or more different antibodies or antigens, and to assay the substances to be tested based on the

  immunological reaction that occurs between these substances.

  According to the present invention, since two or more

  If several substances to be dosed can be dosed at the same time, the time required for dosing can be considerably reduced, when compared with conventional methods which

  involve separate dosages of the substances to be dosed.

  In the accompanying drawings, given only as

example

  Fia 1 to 4 are graphs representing the standard curves of Example 6; Fia 5 to 8 are graphs representing 23 the standard curves of Example 10;

  Fig? is a graph representing the

  bes standards of Example 12; and Fig 10 is a graph representing the

  dilution curves of Example 13.

  The Applicant has discovered that, by the diagnosis

  early cancer thanks to the dosage of tu-

  die, although it is much preferred to do

  separate sages to find out what kinds of tumor markers are present and in what quantities it is practically possible to make a diagnosis based on the total amount of tumor markers, such as

  will prove Examples 2, 4, 6 and 11 described later.

  Based on this discovery, the Applicant has made

  killed intensive research on a process allowing

  to measure the quantity of two or more pre-

  based on a sample, simultaneously dosing the total amount of substances in a single dosing operation As a result, they discovered that one could dosex the total amount of two or more substances to be dosed in one dosing operation based on on an immunological assay principle, assay carried out in

  fixing two or more different antibodies corresponding

  two or more antigens to be assayed, in the same insoluble support or vehicle, or by mixing and using

  two or more insolubilized antibodies-obtained in fi-

  xant of antibodies, corresponding to two or more years

  tigens to be dosed, to separate insoluble supports, without causing any change in the reactivity between each

  antigen to be assayed and the corresponding antibodies The pre-

  sente invention has thus been realized.

  The immunoassay methods on which the assay method according to the present invention is based

  are widely used as physical assay methods

  logic of active substances present in ex-

  very low in samples such as serum, urine, etc., for example hormone levels

  peptides, steroids, proteins, etc., the concen-

  of drugs administered to the human body, etc. Among these techniques, the hemagglutination reaction and the latex agglutination reaction are preferably used, since they have certain advantages such as being simple assay operations, which require little time,

  etc; the immunoenzy-

  radioimmunoassay and immunoassay

  munofluorescence, because these dosages have advantages

  cnmme a great sensitivity, an exce -ente precision, etc.

  The principles of these assay methods will now be briefly described below: 1) Agglutination reaction: When an unknown amount of antigen to be reacted with an antibody reacts

  correspondent linked to a particular support (designated here

  after under the name of solid phase) such as red blood cells, polymer latex, etc., the antigen then binds

  to the antibody on the solid phase depending on its quantity

  tity, which causes agglutination of the solid phase.

  The degree of said agglutination is measured, and the amount of antigen to be determined is determined by comparison with the degrees of agglutination obtained by assaying concentrations

  known of the same substance by analogous orations.

  2) Sandwich method: When an unknown quantity of an unlabeled antigen (anigen to be measured) is reacted with

  a corresponding antibody linked to a solid Dbase (pre-

  reaction), the antigen to be assay binds to the antibody to form an antigen-antibody complex When a given quantity of the same year is reacted: antibody labeled with a labeling agent with said antigen-antibody complex (second reaction ), the marcue antibody binds to the aforementioned complex, but part of the labeled antibody, exceeding the binding capacity of the complex, does not bind and remains in free form Then, the solid phase is separated from

  the liquid phase, the activity of the market agent is measured

  quage in the solid phase or in the liquid phase, and the quantity of unlabeled antigen to be determined is determined on the basis of the standard curve prepared by operations

  analogues using known concentrations of an-

unbranded tigene.

  3) Competitive reaction method: When an unknown quantity of an unlabeled antigen (antigen to be assayed) is reacted in competition as well as a given quantity of a labeled antigen with a corresponding antibody linked to

2531223 1

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  a solid phase, the unlabeled antigen and the antigen mar-

  that respectively bind to the antibody according to their existing quantities, so that the quantity

  of the labeled antigen that binds to the antibody is reverse-

  proportional to the amount of non-antigen

  Then the solid phase is separated from the liquid phase.

  that, the activity of the labeling agent in the solid phase or in the liquid phase is measured, and the amount of the antigen is determined on the basis of the standard curve prepared by analogous operations using

  known concentrations of the unlabelled substance.

  4) Immunometric method: When a quantity is reacted

  unknown amount of unlabeled antigen (antigen to be assayed)

  with a given amount of a corresponding labeled antibody

  then, and then we add the same antigen linked to a solid phase so that it reacts with the labeled antibody which has not reacted, the labeled antibody binds to the antigen

  on the solid phase in an inversely proportional quantity-

  the to the quantity of the unlabeled antigen Then, the solid phase and the liquid phase are separated, the activity of the labeling agent in the solid phase or in the liquid phase is measured, and the quantity of the antigen unmarked is determined based on the standard curve prepared by analogous operations using

  known concentrations of the unlabelled substance.

  The present invention provides a method and a reaction.

  tif to dose the total amount of two at once

  or more substances to be dosed present in a sample

  tillon using the principles of dosing methods

  3 'immunological described above.

  The assay method of the present invention can be applied to any of the conventional immunoassay methods which involve the binding of an antibody or antigen to an insoluble support, by

  example, the hemagglutination reaction, the information reaction

2531223 i

  hibition of agglutination, the reaction reaction method

  petition, the sandwich method, the immunometric method

  that, etc. The process of the present invention is now

  nant described schematically using as sole examples

  pies the agglutination reaction and the sandwvich method. (1) Agglutination reaction: When a sample which is supposed to contain four substances A, B, C and D has to be determined, four different antibodies corresponding to the

  substances to be assayed, namely anti-A antibody, anti-

  anti-B body, anti-C antibody and anti-D antibody, are attached to the same insoluble particulate support, or

  are attached separately to insoluble particulate supports

  individual particles, to prepare a suspension or four different suspensions of insoluble particulate support carrying the four so-called antibodies When the sample is reacted with these suspensions, the substances to be assayed A, B, C and D then bind respectively to the antibodies

  corresponding on the supports, which causes the agglu-

  tination of the particulate supports By measuring the degree of this agglutination, it is possible to measure the total amount of the two or more substances to be measured present in

a sample.

  (2) Sandwich method: When a sample has to be measured

  supposed to contain four substances A, B, C and D,

  tre different antibodies corresponding to the substances to be assayed, namely the anti-A antibody, the anti-B antibody, the anti-C antibody and the anti-D antibody, are fixed to the same insoluble support, or else are fixed separately at

  individual insoluble supports to prepare a sup-

  insoluble port or four different insoluble supports containing the four said antibodies When the sample is reacted with these insoluble supports, the respective substances to be assayed, A, B, C and D, bind respectively

  to the corresponding antibodies on the supports After separation

253,223

  ration of solid bphase, if necessary, we react

  the labeled antibodies obtained by binding a labeling agent

  respective antibody, anti-A antibody, antibody

  anti-3, anti-C antibody and anti-D antibody, with pha-

  solid, so that the respective labeled antibodies

  bind respectively to the substances to be dosed corresponding Don-

  Then the solid phase is separated, then the activity

  total life of the labeling agent bound to the solid phase

  is dosed Thus, one can dose in one go the amount

  totality of two or more substances to be assayed present

in a single sample.

  The insoluble particulate support for use in the agglutination process of the present invention can

  be any of the media conventionally used.

  That is to say bacteria cells, for example red blood cells, etc., latexes of organic polymers,

  such as a polystyrene latex, a polystyrene latex

  incorporating carboxyl groups, a copolymer latex

  styrene-divinylbenzene, a latex of styrene-divinyl polymer

  nylbenzene incorporating hydroxyl groups or groups

  carboxyl, a polyvinyl alcohol latex, a poly latex

  lyacrylate, a vinyl acetate copolymer latex derived from

  vé acrylic, etc, mineral substances like sili-

  ce, carbon black, alumina, etc. can be used

alone or in combination.

  As a substance for the insoluble support in the sandwich method, the method by competition reaction, and the immunometric method, according to the present invention,

  one can use, alone or in combination, any-

  which of the substances used in immunoassays

  classic gics, for example, polystyrene, poly-

  ethylene, polyacrylate, Teflon, paper, glass, agarose, etc. In addition, the shape to be used can be suitably, for example, a sphere, a rod,

  a disc, or some form of container, for example, a

  optical light, test tube, etc., although others

  shapes can also be used.

  Antibodies used in the dosage procedure

  of the present invention can be multi-

  ordinary clonal but the use of mono- antibodies

  clonal gives even better results For example,

  the use of monoclonal antibodies leads to a

  improvement of the sensitivity and accuracy of the assay, a reduction in the reaction time, an improvement in the specificity, the simplification of the assay operations, the exclusion of any non-specific reaction, the exclusion

  any inhibition of the reaction due to the bio-

  logic of the sample, for example serum, urine, etc.

  or similar effects When anti-

  monoclonal bodies in the agglutination reaction, it is necessary to use in combination two or more monoclonal antibodies corresponding to the different antigen sites of a substance to be determined so as to cause

agglutination.

  The method described in "Clinica Chimica Acta" 48, 15 (1973) can be used as the method of attaching one or more antibodies or antigens to an insoluble support. "Journal of Immunology", 116, 1554 (1976); and "Science", 158, 1570 (1967) When it is necessary to fix two

  or several different antibodies to a single carrier

  luble, an antibody solution obtained by mixing the antibodies to be fixed in appropriate proportions, 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 physiological saline buffered with 0.05 M phosphate, p H 6.4, is prepared in advance, then this solution of mixed antibodies is brought into contact with insoluble supports and the reaction is carried out at 30 ° C. for 23 hours After the reaction, the

  supports are washed with physiological saline to pre-

  adorn the supports with fixed antibodies.

  When the anti-AFP antibody, the anti-IICG antibody

  and the anti-CEA antibody must be attached individually-

  ment to separate insoluble supports, said anti-AFP antibodies, anti-HCG antibodies and anti-CEA antibodies are dissolved separately in physiological saline with 0.05 M phosphate buffer, p H 6.4 at concentrations of 0.5 mg / ml,

  0.1 mg / ml and 0.25 mg / ml respectively, then put into con-

  tact with separate insoluble supports, and each mixture

  Age is reacted at 30 56 "C for 2 3 hours.

  Immediately after the reaction, the insoluble supports are washed with physiological saline, to prepare the supports with the respective fixed antibodies. The mixing ratio of the insoluble antibodies thus obtained is appropriately chosen from the range of about 1 10: 1 10: 1 10 well. that it varies according to the quantity of each antibody to

  fix to the insoluble support and the title of each antibody.

  The quantity of each antibody to be attached to the support-

  insoluble is suitably 10 -0.05 mg / ml, preferably 5 0.1 mg / ml, although it varies according to the kind of substance to be assayed, the amount of each substance to be

  dose present in the sample, the title of each year-

body, etc.

  When the assay is performed 9 the opti-

  males are determined experimentally, since the quan-

  tity of sample to be used, the quantity of each

  labeled body or each labeled antigen, and the conditions

  such as reaction time, temperature, etc.

  vary according to the nature of each substance to be dosed, the ti-

  of each antibody used, and the nature of the labeling agent, etc.

  Preparation of labeled antibody and anti

  The labeled gene can be made by a conventional method.

  When the process of the present invention is to

  be implemented based on the competitive reaction

  tion, the labeled antigen to use is the one that will bind

  with the insolubilized antibody competing with the subs-

  tance to be dosed, so that in general, one uses

  cnmme antienne the same substance as the substance to be dosed.

  However, the physiologically active substances, pre-

  in the human body, may exist linked to other

  very biolngic components, or be partially meta-

  bolis-es and so sometimes not being quite the

  same as when they are outside the human body.

  Therefore, if this is the case, a substance which has

  the same reactivity from an immunological point of view.

  that can be used as the same substance to be dosed.

  Although the present invention simultaneously aims at assaying two or more substances, it is also possible to assay a single substance using a single labeled antibody, even when two or more are used

  insolubilized antibodies, unless the substances to be

  will not annoy each other.

  As a marking agent, enzy-

  mes (e.g. peroxidase, p-galactosidase,

  alkaline phosphatase, glucose oxidase, etc.), iso-

  radioactive topes (e.g. 125, H, etc.), subs-

  fluorescent tances (e.g. fluoro isothioxyanate

  rescein, tetramethyl rhodamine isothiocyanate, etc.).

  In the agglutination reaction, by using the support carrying two or more antibodies obtained in the manner described above, it is possible to assay at once

  the total quantity of the two or more substances to be

  ser In the sandwich method and in the competitive reaction method, using two or more labeled antigens or labeled antibodies corresponding to

  two or more antibodies attached to insoluble supports

  sés conne it has been described previously, the total amount of two or more can be dosed at once 33 substances to be dosed In addition, in the sandwich method

  and in the r 6 competitive action method, using

  If you have a single labeled 6 antigen or labeled antibody, you can

  also only dose one substance For example

  A sample diluted to an appropriate concentration is introduced into a test tube where an antibody has been fixed, and the antigen-antibody reaction is carried out. When the reaction is finished, the test tube is washed with

  distilled water, a labeled antibody is added and

  performs the reaction Then, after washing with distilled water

  when tilled, the activity of the labeling agent is measured in the,

  solid phase (test tube) by appropriate means A by-

  from the value obtained, we calculate the quantity 6 of the

  dosing tances present in the sample based on a standard curve obtained by analogous operations

  at known concentrations of the standard substance.

  In this case the standard substance can be a single antigen

  or a mixture of several antigens.

  With the assay method of the present invention, any substance which can be assayed by the

  conventional immunoassay methods Substances

  these particularly important to be assayed are, for example, tumor markers having significant significance

  in the early diagnosis of tumors, the judgment of ef-

  therapeutic effects of the treatments carried out, etc.

  Examples include the carcinoem antigen

  bryonnaire (hereinafter referred to as CEA), l 1 -fetoprotein

  (AFP), human choriogonadotropin (ECG), P 2-micro-

  alobulin (p 2 m), basic fetoprotein (BFP), phos-

  alkaline phatase (ALP), <-glutamyl-transpeptidase (-f GTP), 32glycoprotein associated with pregnancy (SP 1),

  It L "2-glycoprotein associated with pregnancy (SP 3), the pro-

  immunosuppressive acid tein (IAP), it's immunosuppressive 2-macroglobulin, acid isoferritin, fibrinogen, haptoglobin, calcitonin, steroid hormones,

  polyamines, DNA-binding proteins, 1-antitryp-

  sine, pancreatic oncofetal antig O, galactosyl

  transferase (GT-II), etc., as well as many subs-

* tances currently under study In addition, as substances

  having significance in diagnosis and prophyla-

  xie of hepatitis caused by infection with the virus

  from hepatitis a to blood transfusion, there are anti

  hepatitis B virus genes (H Bs, H Bc, H Be) and anti

  non-A non-B hepatitis virus genes.

  Examples of combinations of two or more different substances to be assayed include: (1) AFP, CEA, HCG, (2) SP 3, fibrinogen, (3) AFP, CEA, HCG, SP 3, fibrinogen, (4) fibrinoge & e, haptoglobin, ferritin, (5) haptoglobin, 32 m, 02-macroglobulin immunosuppressive,

  \\ l-antitrypsin, (6) ALP, Y-GTP, GT-II (7) polyamines, hor-

  steroidal mones, (8) H Bs, H Be, and analog combinations

  gues. The embodiment of the present invention makes it possible to reduce the dosing time, while, when

  assay a number of antigens by a conventional method

  that each antigen had to be assayed separately and it had to be

  milk therefore more time In addition, the amount of sample

  necessary has been significantly reduced.

  The present invention is now described in more detail by the following nonlimiting examples:

EXAMPLE 1

  Determination of the total amount of fibrinogen, haotoglobin and úerritine

  a) Production of the latex reagent with fixed antibody.

  Anti-fibrinogen antibody (DAKO Co), anti-haptoglobin antibody (DC, 'O Co) and anti-ferritin antibody

  (DAKO Co) were diluted and mixed in physio-

  looique buffered with 0.05 M phosphate, p H 6.4 (hereinafter referred to as PBS) to obtain concentrations of 5 mg / ml, 1.3 mg / ml and 0.7 mg / ml respectively Was added 0.5 ml of a suspension of 10% polystyrene latex (particles 2531223 i

  uniform latex, Dowv Chemical Co) to 2 ml of the solution

  tion of the previous mixed antibody, and -n was stirred and reacted at 37 ° C. for 2 hours When the reaction was complete, the reaction mixture was cooled with

  3 ice, centrifuged, washed with PBS, and added

  board in PBS containing bo- albumin serum

  wines at 1 5 (referred to below as Drès by BSA), to obtain a

  latex reagent with attached antibody.

  b) Preparation of standard solutions

  Fibrinogen (Sigma Co), haptoglobin (Sig-

  ma Co) and ferritin (Signma Co) have been diluted se-

  facing with PBS containing 1% BSA up to

  centers of 120, 60, 30, 15 and O pg / ml, 12, 6, 3, 1.5

  and O mg / ml and 0.8, 0.4, 0.2, 0.1 and O jug / ml, respectively.

  c) Determination of fibrinogen, haptoglobin and iron

ritine.

  1 of each of the fibrinogen solutions, hap-

  toglobin and ferritin, with their res-

  pectives, produced in b) above were placed on a glass slide, then 50 μl of PBS containing 1 o of BSA and 20 d of the latex reagent with fixed antibody produced in a) above were added. reacted the mixture by shaking for 3 minutes, and the degree of agglutination was observed under the microscope The reaction exhibiting no agglutination was said to be negative (-), and that exhibiting agglutination was said to be positive (+, + + or +++ depending on

  the degree of agglutination); four degrees are thus obtained.

  The sensitivity of the reagent to fibrinogen, haptoglobin and ferritin was 15 pg / ml, 1.5 ug / m J and 0.1 ig / ml

respectively.

EXAMPLE 2

  Determination on serum samples from nalades

  Assays were carried out according to the present invention.

  tion on serum samples obtained from 18 patients suffering from liver cancer, 20 patients suffering 2531223 l from stomach cancer, 20 patients suffering from cancer

  large intestine, 8 patients with lung cancer

  my, 25 patients suffering from various mild illnesses and

  healthy people The operating procedures of the

  wise men were analogous to those of Example 1 c) The re-

  results are shown in Table 1.

TABLE 1

  No lapport Name of disease I Number Dasport judgment of _ + ++ +++ positive of ++ +++ patients (î) Liver cancer 18 1 2 9 6 94 Cancer of the east 20 3 4 8 5 85 Large intestine cancer 20 2 3 7 8 90 Lung cancer 8 O 2 4 2 100 Benign diseases 25 19 5 1 O 24 Healthy humans 20 18 2 OO 10

,; ,, As seen in Table 1, patients can-

  cerous had a high positive ratio, which indicated

  that the utility of the assay method of the present invention

  for: detection of cancer patients.

EXAMPLE 3

  Dose of the total amount of fibrinogen, ha Dtoglobin and ferritin a) Production of Latex with a fixed anti-fibrinogen antibody.

  An anti-fibrinogen antibody (DAKO Co) has been di-

  read with PBS to obtain a concentration of 5 mg / ml.

  0.5 ml of a suspension was added to 2 ml of the diluted antibody.

  polystyrene latex board at% (particular diameter

  medium area 0.48 u, Dowv Chemical), and the mixture was stirred

  ted and reacted at 37 ° C. for 2 hours Once the reaction was terminated, the reaction mixture was cooled with ice, centrifuged, washed with PBS, then suspended in PBS containing 1% BSA to get

  a latex with a fixed anti-fibrinogenic antibody.

  b) -Production of a latex with an anti-haptoglo- antibody

bine fixed.

  An anti-haptoglobin antibody (D Ai O Co) has been di-

  read with PBS to obtain a concentration of 1.8 mg / ml,

  and this solution was used to react with a sus-

  polystyrene latex board has 10 in the same way

  than in a) above to obtain a latex with an anti

anti-haptoglobin body fixed.

  c) Preparation of a latex with a fixed anti-ferritin antibody. 13 Anti-ferritin Ds anticor (D AKO Co) has been diluted

  with PBS Up to a concentration of 1.2 mg / ml, and this-

  the solution was reacted with a suspension of the

  10% v polystyrene tex having carboxyl groups

  tangled (average particle diameter 0.25 Å, Dow Chemical) in the same manner as in a) above to obtain a

  latex with a fixed anti-ferritin antibody.

  d) Production of a latex reagent with attached antibodies.

  The latex with a fixed anti-fibrinogen antibody, the latex with a fixed anti-haptoglobin antibody and the

  latex with an anti-ferritin antibody attached to respective products

  tively in a), b) and c) above were mixed in a ratio of 2: 5: 1 to obtain a latex reagent

with antibodies attached.

  e) Determination of fibrinogen, haptoglobin and iron

ritine.

  Pl of each of the fibrin standard solutions

  gene, haptoglobin and ferritin produced in -l ', xample 1 b) were placed on a glass slide, then 50' 1 of PBS containing 1 G of BSA and 20 μl of the latex reagent with fixed antibodies produced in d) above were added to the standard solution on the glass slide. This mixture was left to react with stirring for 3 minutes, and the degree of action was observed under the microscope. The reaction showing no agglutination was said to be negative (-) and c 2 with an agglutination was said to be positive +: - + or +++ sel'n the degree of agglutination) which gives four degrees The sensitivity of the reagent to fibrinogen, â l khaptoglobin and ferritin was 13 p 9 / nl,

  1.5 g / ml and 0.1 Fg / ml respectively.

  -t O Ek E MPLE 4 Assays on patient serum samples

  Assays were performed using the reagent pro-

  duit in 1 Example 3 on samples of serum obtained

  from 15 patients with liver cancer, 20 patients with stomach cancer, 17

  patients with cancer of the large intestine, 12 to 12 months

  one suffering from lung cancer, 25 patients suffering from

  frant of benign diseases and health The results are given

TABLE 2

  people in the table / ', om of the disease Number of Judgment Report patients _ + ++ positive Liver cancer 15 1 2 7 5 93 Stomach cancer 20 3 4 & 5 85 Cancer of the large intestine 17 2 2 6 7 88 Lung cancer 12 1 2 6 3 92 V; benign diseases 25 20 4 1 O 20 - healthy moms 20 19 1 CO 5 As seen in Cerous report Table 2, the positive high, this

sick can-

which indicates good 2.

2531223,

  the usefulness of the assay method of the present invention

  for the detection of cancer patients.

  EXE Pi-LE 5 Production of purified AFP and Anti-A Fv P 3 antibodies a) Production of purified FP dtli

  16 2 g of crude AFP extract were obtained from

  5 1 ascites of patients suffering from liver cancer by ammonium sulfate salting out Extract

  crude was purified by affinity chromatography in use

  reading 50 ml of Sepharose 4 B with anti-AFP antibody -

  bound rabbit (1 mg antibody / ml Sepharose) to obtain

nir 924 mg of purified AFP.

  b) Production of anti-AFP monoclonal antibody pg of purified AFP produced in a) above as well

  13 that Freund's full adjuvant (FCA) was administered

  very subcutaneously in a BALB / c mouse.

  nistrations were repeated four times a week

  interval, and four days after the last administration

  tration, the spleen was isolated, and the spleen cells

  were collected spleen cells were la-

  ves with modified 5 EM medium from Dulbecco (designated

  after by D-ME), 1x10 O cells were counted and mixed

  diapered with 1 x 10 mouse myeloma cells

  (P 3-NSI / l-Ag 4-1) They were subjected to a cel-

  in a ml of D-MEM containing 42.5 of polyethylene

  lene glycol 1540 and 7.5% dimethyl sulfoxide at 37 C

  for 1 minute HAT medium was added to these cells

  (RPMI-1640 medium containing hypoxanthine, amino-

  pterine, thymidine and 10% fetal serum from bo-

  wine) to a volume of 20 ml, then the mixture of cellu-

  was distributed in the wells of a 96-well microplate, at a rate of 0.2 ml per well, and cultured for 2

  weeks after which the activity of the antibody in the over-

  swimming culture wells where there has been proliferation

has been measured.

  Then, the cells from the wells having an activity were collected and added to 40 ml of RPMI-1640 medium containing 10% of fetal bovine serum and

  thymus cells of a BALB / C mouse This suspension

  cells were distributed in two 96-well microplates, and cultured for one week to obtain

  9 hybridoma clones producing an anti-AFP antibody.

  They were transferred to large-scale cultures, and one liter of each culture supernatant obtained was subjected to affinity chromatography using 50 ml of Sepharose 4 B with bound purified AFP (0.5 mg AFP / ml of Sepharose) to obtain 4.2 to 11.6 mg of monoclonal antibodies, respectively. The respective antibodies were

designated by Lot N 1 9.

  c) Identification of antigen recognition sites

  i) Production of test tubes with anti-antibody

AFP fixed.

  Volumes of 1 ml PBS containing 0.5 mg of

  the monoclonal anti-Av P antibody Lot N 1 9 were res-

  pectively added to separate polystyrene test tubes

  oares having been washed with PBS, and each test tube was incubated at 37 ° C for 3 hours to facilitate

  attachment of the antibody to the internal surface of the test tube

  sai After the reaction, the test tubes were washed with PBS to obtain antibody test tubes

monoclonal fixed.

  ii) Production of the anti-AFP antibody labeled with

an enzyme.

  mg of horseradish peroxidase (Behringer 1 year- heim Co, Quality I; hereinafter referred to as HRPO) were dissolved in 1.0 ml of sodium bicarbonate buffer

  0.3 M, and to this solution was added 0.1 ml of 1-fluoro-

  2 4-dinitrobenzene at 1% o in ethanol and the mixture

  was reacted for one hour Then 1.0 ml of u

  a 0.06 M sodium periodate solution was added to the mixture, the reaction took place for 30 minutes, then 1.0 ml of a 0.16 hi ethylene glycol solution was added,

  and the reaction was allowed to continue for one hour.

  The reaction mixture was dialyzed against a 0.01 M sodium carbonate solution, p H 9.5. To this was added

  te 5 mg dialysate solution from each of the 9 lots of anti

  anti-i FP rims produced in b) above, and each mixture was allowed to react at ambient temperature for 3 hours Then, 5 mg of sodium borohydride was added,

  and the reaction was allowed to continue overnight.

  The reaction mixtures were respectively dialyzed against 0.01 N PBS, p H 7.2 to obtain each antibody.

anti-AFP labeled with HPO.

  iii) Identification of sites of recognition of an-

elder.

  To each of the test tubes with fixed anti-A Fr P antibody 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 AP produced in a) above and

  0.4 ml of a hundredth dilution of each anti-

  : P labeled with HRPO produced in ii) above, and each reaction took place for 30 minutes When the reaction was complete, each test tube was washed with a washing solution, 0.5 was added ml of an enzyme substrate solution containing 20 mg / dl o-phenylenediamine and 6 m M hydrogen peroxide, and the reaction took place for 30 minutes, 2 ml of 1N hydrochloric acid were

  added to stop the enzymatic reaction, and the absorption

  bance of the reaction mixture at 492 nm was measured Les

  results are presented in Table 3, the combinations

  sounds carrying out the colored reaction being noted + and those

  not realizing it being noted -.

  -20 TAl 3 LEAIJ 3 Antibody labeled with 11 HRPO

1 2 3 4 5 6 7 8 9

u 1 + + + +

_ 4 + _ + + +

3 + + + +

vs

4 + + + + + + -

H _

+ + +

6 + + + + + ++ +

O 7 + + + +

8 + + + + + +

9 + + + + + + -

  According to the difference in recognition sites

  antigen, the 9 lots of anti-AFP 1-5 1 monoclonal antibodies were classified into 3 groups, that is to say the first group consisting of Lots N 1, 2, 3, 5 and 7, the

  second group consisting of Lot N 6 and the third group

  eg consisting of Lots N 4, 8 and 9 These groups were

  named anti-AFP antibodies (A), (B) and (C) respectively.

  Among them, (A) will be used as an insoluble antibody.

  bilised and (C) will be used as a labeled antibody

in the following examples.

PLE EXE 6

  Production of purified CEA and anti-CEA antibodies

a) Production of purified CEA.

  g of cancerous tissues of the large intestine were minced and, after the addition of 100 ml of distilled water, homogenized in a homogenizer The same volume of 1.2 M perchloric acid was added to this suspension and

  the mixture was extracted with stirring for 30 minutes.

  The centrifuged supernatant was dialyzed against distilled water.

  tilled to obtain a crude extract of CEA.

  This crude extract was concentrated to 10 ml and subjected to gel filtration using Sepharose 4 B balanced with physiological saline and the first protein reaction was collected. Then, the fraction

  was again subjected to gel filtration in use

  reading Sephadex G-200 balanced in the same way and the second protein fraction was collected, which was then concentrated to 2 ml to obtain 135 g of

Purified CZA.

  b) Production of monoclonal anti-CEA antibody Eight clones of a hybridoma producing an anti-CEA Ds anticor were obtained from the purified CEA produced in a) above by procedures analogous to those of Example 5 b) To immunize the mice, we have

  used with each administration 30 g of purified CEA.

  1 x 10 hybridoma cells were inoculated intraneritoneally into a BALB / c mouse

  had administered intraperitoneally 0.5 ml of taken

  tane (2, 6, 10, 14-tetramethylpentadecane; a product of Wako Pure Chemicals, Co, Ltd) Two weeks later, ascites were collected Each ascites was subjected to a

  chromatography on DEAE-cellulose balanced with a tam-

  0.01 M phosphate pon, p H 7.0, to obtain a monoclonal anti-CEA antibody in the non-adsorbed fraction As

  result of the identification test of recognition sites

  antigen according to Exemole 5 c) antibodies

  were classified into three groups, each consisting of

  no of 5 lots, 2 lots and 1 lot, which have been named respec-

  anti-CEA antibodies (A), (B) and (C) Among them,

  the anti-CEA antibody (A) will be used as an insoluble antibody

  lubricated and the anti-CEA antibody (B) will be used as

  antibody labeled in the following examples.

EX El 'PLE?

  Preparation of HCG-E and anti-HCG- antibodies.

  a) Production of a HCG-p subunit One gram of HCG (2,000 IU / mg) was dissolved in 2 ml of 0.025 M phosphate buffer, p H 5, -6 and subjected to

  chromatography on 3 9 of DE-_Sephadex-A-50 equili-

  break with the same buffer The fraction eluted with 0.05 M phosphate buffer, p H 5.6 was collected and dialyzed against distilled water to obtain 308 g of purified HCG, which was then lyophilized 300 mg of Durified HCG were dissolved in 10 ml of 10 M urea (OH 4.5) and the reaction

  took place at 40 C for one hour, followed by a chroma-

  tograohie using 2 g of DAE-Se Dhadex A-50 balanced with a solution containing 0.03 M glycine and urea 'M The fraction eluted with a solution containing

  0.2 M glycine, 1 M Na Cl and 8 M urea was dialy-

  against physiological serum to obtain 147 mg of

HCG-3 subunit.

  b) Production of anti-HCG-3 antibodies Eleven batches of hybridomas producing anti-HCG-J 3 antibodies were obtained from the HCG-3 subunit

  produced in a) above by analogical procedures

  gues to those of Example 5 b) Identification of sites

  antigen recognition was done on the anti-

  bodies obtained from these batches, according to the operating modes

  roofs of Example 5 c), which made it possible to classify them into 2 groups comprising 8 batches and 2 batches respectively, and to designate them respectively by anti-I 4 CG (A) antibody and anti-HCG-3 antibody ( B) The anti-HCG-F antibody (A) will be

  used in the following examples as an insoluble antibody

  and the anti-HCG-3 antibody as the labeled antibody.

  When these antibodies have been examined for their cross-reactions with luteinizing hormone (LH) using the

  above combination, reactivity was found inferior

  less than i, the reactivity for HCG being taken for 100%.

3 N EXE-MIPLE 8

  Determination of the total amount of AFP, CEA and HCG a) Production of a reagent consisting of a test tube

with antibody attached.

  To polystyrene test tubes was added 1 ml of a solution containing 0.1 mg / ml of anti-AFP antibody: ionoclonal, 0.5 mg / ml of anti-CEA monoclonal antibody and

  Q, 25 mg / ml monoclonial anti-HCG-n antibody produced res-

  pectively in examples 5, 6 and 7, and each tube has

  test was incubated at 37 ° C for 3 hours When the

* cuoation has been completed, each test tube has been washed with

  P 13 S, to obtain the reagents made up of

sai with antibodies attached.

  b) Preparation of standard solutions 1 AFP produced in Example Sa), the CA produces

  in Example 6 a) and HCG (HICG Mochida, Mochida Pharma-

  ceutical) were diluted with PBS containing 1% of BSA nour prepare solutions of 80, 40, 20, 10, 5 and O ng / ml,, -0, 20, 10, 5 and O ng / ml and 80, 40, 20, 10, 5 and O

miui / ml respectively.

c) Determination of AFP, CEA and HCG

  To reagents made up of test tubes with anti

  fixed bodies produced in a) above were added Portions of 0.1 ml of standard solutions of AFP, CEA and HCG with their respective concentrations, followed by the addition

  0.4 ml portions of PBS containing 1% BSA, and each

  the reaction took place at room temperature for 2

  hours When the reaction has been completed, the test tubes

  sai were washed with distilled water Besides,

  the antibodies labeled with the enzyme produced in the Examples

  plates 5, 6 and 7 were diluted and mixed in PBS con-

  holding 1% BSA to obtain a solution containing a

  1/2000 dilution of anti-AFP antibody labeled with

  zyme, a 1/800 dilution of anti-CEA antibody labeled pu

  With the enzyme and a 1/500 th dilution of the anti-

  Enzyme-labeled HCG 0.5 ml portions of said mixture were added to the previous test tubes, and each

  reaction took place at room temperature for 2 hours.

  When the reaction was complete, the test tubes were washed with distilled water, portions of a substrate solution were added (6 m M / 1 of hydrogen peroxide and 2 to m M / 1 o-ph nylnediamine in PBIS), and each

  reaction took place at room temperature for 30 minutes

  away from light Then 2 ml of chlorhy-

  1 1 dric were used to stop the reaction, and the absorbance of the reaction mixture was measured at a 492 nm wavelength. Dosages were also carried out for the comparison, in the case where the antibody

  marked were not mixtures but reacted separately.

  The results are summarized in Figures I f 4 The combinations

  It sounds of the insolubilized antibody and of the labeled antibody used in the respective figures are presented in

Table 4.

TABLE 4

  From the previous results, it can be seen that since AFP, CEA and HCG do not cross-react with each other, they can be dosed

  individually using the labeled antibodies res-

  even if these substances are present as a mixture, and even when the labeled antibodies are

  used as a mixture, only antigens and an-

  respective corresponding antibodies react and this reaction

  activity is not influenced by the presence of other

  tances. Anti-insolubilized antibody Labeled antibody Anti-AFP antibody, Fig 1 anti-CEA antibody & Anti-AFP antibody, anti-HCG antibody in anti-CEA antibody mixture & in anti-HCG antibody mixture Fig 2 antiHCG antibody Anti-AFP antibody

mixed alone -

  Fig 3 Antibody anti-CEA alone Fig 4 Antibody anti-HCG alone

EXAMPLE 9

  Assay on patient serum samples

  Serum samples taken from 10 suffering patients

  frant of liver cancer, 10 patients suffering from a

  stomach cancer, 10 patients with fat cancer

  intestine, 10 patients suffering from various benign diseases

  healthy people and 10 hpm were dosed for the total amount of AFP, CEA and HCG Each dosage was

  performed using 0.1 ml standard solution or se-

  rum to be tested by procedures similar to those of Example 8 c) The results are expressed by measuring

  absorbance directly at 492 nm and also in con-

  vertie in CEA, obtained by applying said value of the ab-

  sorbance as an experiment to the standard curve of the CEA.

  For comparison, AFP, CEA and HCG were also measured

  res individually on the same samples

  results are shown in Table 5.

2551223 It

TABLE 5

  Name of present invention Reference disease Absorbance Converted value AFP CEA HCG 492 nm to ng / ml ng / ml mui / 'ml CEA ng / 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 J 3 2.2 2.0

  Cancer 3,205 147.5 158.7 0.2 liver 2.0 0.21 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 1-5.0 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

  Cancer 0.492 19.8 1.1 1.7 21.1 0.501 16.8 1.0 17.2 0.8 Stomach 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

03.260 4.7 2.0 1.0 4.0

  TABLE 5 (continued) Name of the present invention Illness-related illness Absorbance Value 492 nmconverted AFP CEA H {CG 492 'in ng / ml ng / ml mui / mil CEA ng / ml

0.788 4 Y 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

  Cancer 0.388 10.1 1.95 11.3 0.8 large $ 0401, 30 31 intestine 047148 12 1.32

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 V 521.8 2.0

0.231 4.0 1.3 0.3 3.0

0.219 3.8 1.3 0.2 1.9

0.320 6.9 3.1 3.2 2.22

0.301 6.0 2.1 3.1 2.5

  Diseases 0.371 9.8 2.2 6.1 -1.1 mild 0.333 8.5 2.5 1.8 3.5

0.261 4.9 0.8 2.1 3.3

0.321 7.1 2.9 292 2.2

0.271 S Pl 0.8 3.1 2.8

0.270 5.1 4.1 0.9 1.0

  TABLE 5 (continued) Name Present invention Disease reference Absorbance Value 492 nm converted AFP CEA HCG to ng / ml ng / ml mui / ml CEA ng / ml

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

  Humans 0.259 4.7 0.7 2.8 3.3 good 0.268 4.8 2.0 2.1 2.0 health 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

i,

Table 6

  of positivity in 'below presents the patients suffering from percentage each disease, obtained in the dosages of a single parameter, when

  assumes that diagnostic benchmarks for pre-

  suspected cancer are 10 ng / ml or more for AFP, 5 ng / ml or more for CEA and 5 mui / ml or more

for 1 HCG.

  Table 6 also presents the percentages of positivity in patients suffering from each disease in the dosages of Table 5 above, o, when one

  dose the total amount of the three substances according to the pro-

  c 4 dice of the present invention, the absorbance at 492 nm of 0.350 or more or the value converted into CEA of 8.5 ng / ml

  or more are considered positive for cancer.

TABLE 6

  Name of Present AFP CEA HCG One or more

  disease invention markers Liver cancer 80% 80% 20 O% 80% Stomach cancer 60 10 20 30 60 Cancer of the large intestine 60 10 60 O 60 Benign diseases 10 O 10 O 10 Healthy humans OOOOO In addition, when the positivity percentages

  in patients with each disease are calculated as suppo-

  health that a patient is "positive for cancer" if at least one of the three tumor markers measured individually exceeds the standard values, these percentages of positivity,

  which are also included in the right column of the Ta-

  bleau 6, were the same m 9 as the percentages de'positi = vity for cancer in the case where the three markers of

  tumors were measured in total quantity (second co-

  lonne of Table 6) This indicates that during a cancer diagnosis, it is not necessary to assay markers

  tumors individually, but in practice a diag-

  nostic is possible from the measured value of the

total quantity.

- EXAMPLE 10

  Determination of the total amount of AFP, CEA and HCG

  a) Production of a reagent consisting of a pearl of polys-

tyrene with antibody attached.

  The monoclonal anti-AFP antibody, the anti-

  Monoclonal CEA and monoclonal anti-HCG antibody produced in Examples 5, 6 and 7 respectively, were diluted

  at concentrations of 1.0, 0.8 and 0.5 mg / ml respectively-

  Polystyrene beads (2.5mm diameter) were soaked in each solution, and each mixture was reacted at 37 OC for 3 hours. When the reaction was complete, the beads were washed with PBS to prepare reagents consisting of polystyrene beads with antibody attached, each set of reagent being

  consisting of 3 pearls each carrying different antibodies

  rents. b) Determination of AFP, CEA and HCG 0.1 ml portions of the solutions were added to test tubes (internal diameter mm, length 60 mm)

  AFP, CEA and HCG standards with their res-

  pectives prepared in lt Example 8 b '), then was added portions of 0.4 ml of PBS solution containing 1% BSA and a reagent set consisting of three different beads of polystyrene with fixed antibody produced in a) ci above After stirring, the reaction took place at room temperature for 2 hours When the reaction was complete, the polystyrene beads were washed

  with distilled water Furthermore, the antibodies

  ques the enzyme produced in Examples 5, 6 and 7 were diluted and mixed in PBS containing 1% o of BSA to obtain a solution containing a dilution to 1/2500 th of the anti-AFP antibody labeled with the enzyme, a 1/1000 dilution of the anti-CEA antibody labeled

  the enzyme and a 1/650 dilution of the anti-

  HCG labeled with the enzyme 0.5 ml portions of the said mixture

  ge were added to the previous beads, and each re-

  action took place at room temperature for 2 hours.

  When the reaction has been completed, the polysty-

  were washed with distilled water, added

  0.5 ml portions of a substrate solution (6 m M / l-

  hydrogen peroxide and 20 m M / 1 o-phenylenediamine

  in PBS), and each mixture was reacted

  room temperature for 30 minutes away from light

  Then, 2 ml of 1N hydrochloric acid was added to stop the reaction, and the absorbance was measured at a wavelength of 492 nm. For comparison,

  assays were also carried out in the event that the an-

  3 tagged antibodies were not mixed but separated The results are summarized in Figures 5 8 o Combinations

  insolubilized antibody and labeled antibody used

  read in the respective figures are presented in the

Table 7.

TABLE 7

  Antibody insolubilized Labeled antibody Anti-AFP antibody Fig 5 anti-CEA antibody & Mixture of three beads anti-HCG antibody

in mixture -

  Pearl with antibody Anti-AFP antibody

Fig 6 anti-AFP fixed alone.

  pearl with fixed anti-CEA antibody and pearl with fixed anti-HCG antibody Fig 7 Anti-CEA antibody alone Fig 8 Anti-HCG antibody

only -

  According to previous results, in the same way

  re that in Example 8, it can be seen that, since AFP, CEA and HCG do not cross-react with each other, they can be assayed individually using the respective labeled antibodies even if these

  substances are present as a mixture, and that mix-

  me when the labeled antibodies are in the form of a mixture

  ge, only the corresponding antigens and antibodies remain

  pective react and this reactivity is not influenced

  by the presence of other substances.

EXAMPLE it

  Assay of patient serum samples Serum samples from 10 patients suffering from liver cancer, 10 patients suffering from stomach cancer, 10 patients suffering respectively

  large bowel cancer, 10 patients with ma-

  benign ladies and 10 healthy humans were assayed for the total amount of AFP, CEA and HCG Each assay was done using 0.1 ml of standard solution or serum to be tested by procedures analogous to those

  of Example 8 c) The results are expressed by the me-

  direct absorbance at 492 nm and also by the

  value converted into CEA obtained by applying said value

  their absorbance as an experiment at the state curve

  lon CEA For comparison, AFP, CEA and HCG were also

  dosed individually on the same samples.

  The results are presented in Table 8.

TABLE 8

  Name of Present Invention Reference aladia Absorbance Value A CA HC disease 492 nm converted AF Pm ng / m mui in CEA n / in / imi ne / mi mil

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

  Cancer 0.518 21.6 13.8 7.2 1.6 Liver 0.197 4, q 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, q 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

  Cancer 0.481 19.6 5.2 0.3 15.9 stomach 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 12.10 3.5 0.2 8.1

  TABLE 8 (continued) Name of the present invention Reference disease Absorbance AFP value CEA HCG 492 nm converted ng / ml ng / ml mui / ml to CEA ___________ n g / m l_ _ _ _ _ _ _ _ _ _ _

$ 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

  Cancer 0.785 35.9 1.6 35.0 1.1 of the large 0562,) 1, e intestine 051215461, 1,

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 e 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 0.4 111

0.230 6.1 2.3 0.8 3.0

0.249 7.1 3.4 1.4 2.15

  Diseases 0.177 3.2 1.7 0.3 1.9 mild 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 1.4 111

0.203 4.6 2.8 1.8 1.2

- TABLE 8 (continued)

Table 9

  positivity in patients below presents the percentages of each disease obtained in single parameter assays when it is assumed that the diagnostic standard values for the suspected presence of cancer are 10 ng / ml or more for 1 i AFP , 5 ng / ml or more for CEA and 5 mui / ml or more for lt HC Go Table 9 also presents the percentages of positivity in patients of each disease in the dosages of Table 8 above, in which, when the total quantity of the three said substances is measured according to the method of this

  invention, the absorbance at 492 nm of 0.350 or more or its

  their converted to CEA of 8.5 ng / ml or more are considered

as "positive for cancer".

  Name of Present Invention Reference disease Absorbance AFP value CEA HCG 492 nm converted ng / m ng / ml mui / in CEA ng / ml ng / ml mui / ml in CEA ng / ml

0.155 290 1.2 0.2 190

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

  Human 0, t 61 2.3 1.0 0.3 1.0 in 0.192 4.0 2.1 0.2 2.0 good O 0181 3.4 1.0 1.6 1.3 health 0.155 2, 0 0.8 0.3 1.0

0.157 2.1 0.8 0.4 1.4

0.155 2.0 0.6 093 1.4

10.

TABLE 9

  i I l I Name of the present AFP CEA HCG One or more inven marking diseases liver cancer 80% 80% 20% O% 80 o Stomach cancer 60 10 20 30 60 Large intestine cancer 60 10 60 O 60 Diseases b 6 vines 10 O 10 O 10 Healthy humans OOOOO In addition, when the percentages of positivity

  in patients with each disease are calculated as suppo-

  health that the patient is "positive for cancer" if at

  minus one of three individual tumor markers dosed-

  ment exceeds the standard values, these percentages of posi-

  tivity, which are also presented in Table 9, were the same as the positivity percentages for cancer

  in the case where the three tumor markers 6 were measured

  res in total quantity This indicates that during a diagnosis

  tic of cancer, it is not necessary to dose the mar-

  tumors individually, but in principle a diagnosis is possible from the measured value of

the total amount.

* EXAMPLE 12

  Determination of the total amount of fibrinogen and SP 3 a) Production of reagent consisting of Sepharose 4 B with antibody fix 6

  The anti-fibrinogen antibody (DAKO Co) and the anti

  anti-SP 3 bodies (DAKO Co) were respectively diluted with 0.1 M sodium bicarbonate buffer, p H 8.3 for

  obtain concentrations of 5 and 3 mg / ml respectively.

  Dilutions were added to 5 ml portions.

  ves 5 ml portions of Sepharose 4 B activated at CN Br (Pharmacia Co) and the reaction took place at room temperature.

  biante during 2 hours When the reaction was finished

  The reaction mixture was washed with 0.1 M acetate buffer containing 0.5 M sodium chloride, then with PBS, which gave Sepharose 4 B with the anti-antibody. -fibrinogen fixed and Sepharose with the anti-SP antibody fixed They were mixed in a 1: 1 ratio to produce a reagent consisting of

  Sepharose 4 B with attached antibodies.

  b) Preparation of standard fibrinogen solutions Fibrinogen (Sigma Co) was diluted with PBS containing 1% BSA to prepare standard solutions

  80, 40, 20, 10, 5 and O, g / ml respectively.

  c) Preparation of standard solutions of SP 3 According to the method of Hans Bohn et al (Blut Band 33, 377-378, 1976), 5 kg of placenta were extracted with

  physiological saline, fractionated using Riva-

  nol and ammonium sulfate, and purified by chromatography

  affinity phy using 50 ml of Sepharose 4 B with fixed antiSP 3 antibody (1 mg of antibody / ml of Sepharose) to obtain 3.7 mg of purified SP 3 This purified SP 3

  was diluted with PBS containing 1 BSA ode for preparation

  rer standard solutions of 80, 40, 20, 10, 5 and O g / ml respectively.

  d) Production of anti-fibrinogen antibodies labeled with

  zyme and enzyme labeled anti-SP antibody The enzyme labeled antifibrinogen antibody and enzyme labeled anti-SP 3 antibody were produced from the anti-fibrinogen antibody and anti-SP 3 antibody by procedures analogous to those of

Example 5 c) ii).

  e) Determination of fibrinogen and SP P 3 0.1 ml portions of standard solutions of fibrinogen and SP 3 were added to separate test tubes with their respective concentrations produced in b) and c) above, followed by the successive additions of portions of 0.4 ml of PBS solution containing 1% of BSA and of 0.2 ml portions of the reagent consisting of Sepharose 4 B with fixed antibody produced in a) above, and, after stirring, each reaction took place at room temperature

  for one hour After the reaction, the contents of the

  test bags were centrifuged and washed with distilled water.

  In addition, the labeled anti-fibrinogen antibody

  has the enzyme and anti-SP 3 antibody labeled with the enzyme pro

  duit in d) above were mixed and diluted to con-

  maintain a 1/1500 th dilution of the anti-fibri-

  nogen labeled with the enzyme and a 1/10 dilution of the anti-SP 3 antibody labeled with the enzyme Portions of

  0.5 ml of the mixture was added to the previous test tubes

  teeth, and the mixture of each test tube was reacted at room temperature for one hour. When the reaction was complete, the contents of the test tubes were centrifuged and washed with distilled water, and after addition 0.5 ml portions of a solution of

  substrate (6 m M / 1 of hydrogen peroxide and 20 m M / 1 of o-

  phenylenediamine in PBS), each mixture was reacted at room temperature for 30 minutes under cover

  light Then 2 ml of chlorhy-

  dric 1 N to stop the reaction, and the absorbance of the resulting solution was measured at a wavelength of

  492 nm The results are presented in Figure 9.

EXAMPLE 13

  Determination of the total amount of AFP, CEA and HCG a) Preparation of the LAFP standard solutions produced in Example 5 a), the CEA produced in Example 6 a) and 1 HCG (HCG Mochida) were diluted

  with PBS containing 1% BSA to prepare solutions

  80 ng / ml, 80 ng / ml and 80 mui / ml respectively, and these solutions were mixed in various proportions presented in Table 10 to prepare 5 different mixed solutions o

TABLE 10

  Number of the mixed solution AFP CEA HCG

1 1 1 1

2 5 1 1

3 I 5 1

4 1 1 5

3 2 1

  Each of the above mixed solutions 1 5 was diluted to half, quarter and eighth with

  PBS containing 1 5 of BSA to prepare the standard solutions

  b) Determination of AFP, CEA and HCG Using 0.1 ml portions of the standard solutions produced in a) above and the mixtures of the three

  different labeled antibodies, two assays were performed

  not analogously to that of Example 19 c) 0 The respective standard curves are presented in FIG. l Oo Even when the standard solutions containing AFP, CEA and HCG in mixture were used, standard curves similar to those obtained were obtained of figure 1 This indicates that, even when AFP, CEA and HCG are present in mixture, they can be assayed individually and therefore

  the dosage value of their total quantity can be obtained

  naked by the method of the present invention.

Claims (12)

R E V E N D I C A T I O N S   1 An immunoassay method characterized in that a sample containing two or more reacts   several substances to be dosed with two or more anti   body or antigens insolubilized simultaneously and to be assayed   said substances based on an immune reaction   logic occurring between said substances and   two or more insolubilized antibodies or antigens.   2 assay method according to claim 1, ca-   characterized in that the two or more insolubilized antibodies or antigens are attached together to an insoluble support.   3 assay method according to claim 1, ca-   characterized in that the two or more antibodies or an-   insolubilized tigens are attached respectively to sup- separate ports.   4 The assay method according to claim 2, ca-   characterized in that the insoluble support is chosen from   red blood cells, polymer latex, car black   bone and an inner surface of a reaction vessel.   Assay method according to claim 3, characterized in that the insoluble supports are chosen from red blood cells, a polymer latex and black of carbon.   6 Dosing method according to any one of   Claims 1 to 3, characterized in that the reaction   immunological is an agglutination reaction or a   agglutination inhibiting action.   7 Dosing method according to any one of   Claims 1 to 3, characterized in that the method   immunoassay is an enzyme immunoassay,   a radioimmunoassay or an immunoassay   rescence. 8 assay method according to claim 7, characterized in that the immunological assay method   is also chosen from a sandwich method, a   competition reaction method and an immuno- method   metric. 9 Dosing method according to any one of   claims 1, 2, 3, 6, 7 and 8, characterized in that   said antibodies are tu markers antibodies   die. Dosage method according to any one of   claims 1, 2, 3, 6, 7, 8 and 9, characterized in   that the antibodies are monoclonal antibodies.   11 An immunoassay reagent designed to react with a sample containing two or more   substances to be determined by an immunological reaction, characteristic   terized in that it comprises a mixture of two or more insolubilized antibodies or insolubilized antigens * 12 Assay reagent according to claim 11,   characterized in that the two or more antibodies in-   solubilized or insolubilized antigens are fixed together to an insoluble support.   13 Assay reagent according to claim 11,   characterized in that the two or more antibodies in-   solubilized or insolubilized antigens are fixed respec-   tive to separate insoluble supports.   14 Assay reagent according to claim 12, characterized in that the insoluble support is chosen from red blood cells, a latex of high molecular weight, carbon black and an internal surface of a container of reaction.   Assay reagent according to claim 13, characterized in that the insoluble vehicles are chosen from: red blood cells, polymer latex and car bone black. 16 Assay reagent according to any one of   claims 11 to 13, characterized in that it is designed   to react in an agglutination reaction or a reaction agglutination inhibition.   17 Assay reagent according to any one of   claims 11 to 13, characterized 6 in that it is designed   to react in a chosen immunoassay method   sie among an immunoenzymatic assay, a radioim-   munological and immunofluorescence assay.   18 Assay reagent according to claim 17,   characterized in that said immunoassay method   that is still chosen from the sandwich method, the   competitive reaction method and the immuno- method   metric. 19 Assay reagent according to any one of   claims 11 to 13, characterized in that said anti   bodies are antibodies to tumor markers.   20 Assay reagent according to any one of   claims 11 to 13, characterized in that said an-   antibodies are monoclonal antibodies.