FR2548901A1 - Diagnosis of human adenocarcinoma by detecting specific determinants - Google Patents

Abstract

Diagnosis comprises detecting an AC-associated antigen using as reagent (a) anti-K antibody (Ab1) prepd. by immunising an animal with K antigenic determinants free of Pk or Pn determinants or (b) anti-Pk antibody (Ab2). Ab1 detects only the K determinants specific for AC while Ab2 detects p3Pk determinants associated with K or AC. The process can be applied to a sample taken from a human patient or used in vivo the cells of the tumour itself. Ab1, Ab2 and also anti-Pn antibody (Ab3), esp. where these are monoclonal, and including the conjugates of Ab1 and Ab2 with anti-tumour or cytotoxic agents. The K, Pk and Pn antigenic determinants are also new (Pn are present in normal plasma and serum and give a cross-reaction with Pk).

Description

New antigenic determinants related to
adenocarcinoma and antibodies specific to them,
processes for preparing them and combinations of reagents
relating thereto
The present invention relates to novel adenocarcinoma-related antigenic determinants, to antibodies specific thereto, and to methods of diagnosing human adenocarcinoma based on the detection of adenocarcinoma. antigenic determinants in biological fluids, in extracts or sections of tissues and in vivo on cells or tumor masses.

 The invention also relates to the use of specific antibodies in the therapy of adenocarcinomas to reach cells or tumor masses by antitumor drugs or other cytotoxic agents. It is safe to say that, in the case of tumoral pathologies, it is possible to isolate, from the same tumoral masses or biological fluids, substances associated with these pathologies. Many of these substances have more marked antigenic properties in animals. that in humans some of them, generally called "tumor markers", are oncofoetal antigens. The presence of an antigen which is said to be mainly associated with adenocarcinomas, and especially with their metastases, has been demonstrated by Gold et al, J. Expt. Med. 121: 439-462 (1965).

This antigen, known as carcinoembryonic antigen (CEA) can be assayed both in biological fluids and in extracts or tissue sections by various known methodologies, for example those reported in US patents. of America
Nos. 3,663,684 and 3,697,638; Ann. of Clin. and Lab. Science 4, 5, 357, 1974; Clin. Chem. 19, 10, 1973; Cancer 34: 15041509, 1974; Clin. Res. 19: 143, 1971; Proc. Natl. Acad. Sci.

USA 64: 161-167, 1969; Cancer 37: 62-81, 1976; Scand. J.

 Immunol., Vol. 7 Snppl. 6, 1978; Cancer 45: 1243-1247, 1980 and J. Natl. Cancer Inst. 57, 1, 1976.

 The many studies that have been done in this area, including for example the above references, however, revealed the very low specificity of the CEA, because it has been demonstrated not only in the case of tumor pathologies but also in the presence of different pathologies, that is to say non-tumorous, such as for example many inflammatory degeneration processes, and even in healthy donors. In addition, biochemical studies [Urba R .; Alpert E., Isselbacher K.J., Proc.

 Natl. Acad. Sci. U.S.A. 72, 4602-6 (1975)] have revealed differences between CEAs obtained from different laboratories, with the result that different clinical results are obtained when the same techniques are used with substances of different origin.

 It has now been discovered that it is possible to obtain from human adenocarcinoma metastases, as well as from all human adenocarcinomas, for example gastric, mammary and thyroidal adenocarcinomas, antigenic determinants (K) which are "specific" to adenocarcinomas, and antigenic determinants (pk) which are "associated" with the K determinants on adenocarcinomas.

While the antigenic determinants pk show a very strong cross-reaction with the antigenic determinants (Pn) found in the plasma or serum of normal donors, the antigenic determinants K show no cross-reactivity with the antigenic determinants Pn. This is the mixture of said antigenic determinants K and R and perhaps some other unknown antigens that were previously known as CEA.

 Using antigenic determinants K and the aforementioned antigenic determinants Pk and Pn, anti-K, anti-Pk and anti-Pn specific antibodies are obtained according to the invention.

 The use of the antigenic determinants K and Pk and anti-K and anti-Pk Pk antibodies which are specific to them in known immunological methods, such as, for example, the radioimmunoassay, the enzymoimmunoassay, the immunofluorescence, the Immunoluminescence, and many others, can detect and quantify exclusively the antigenic determinants K which are specific to adenocarcinomas, without interference deriving from pn antigenic determinants of plasma or normal serum, or, where appropriate, the antigenic determinants pk which are associated with the determinants K on adenocarcinomas. The above determination can be done either qualitatively or quantitatively or semi-quantitatively.

The subject of the present invention is therefore an improved method for diagnosing human adenocarcinomas by determining the antigen substance related to adenocarcinoma, either in a sample taken from a human patient, for example in vitro on biological fluids or extracts or tissue sections, or in vivo on the cells or tumor masses, the improvement consisting in determining, by means of specific anti-K or anti-pk antibodies, exclusively K antigen determinants which are specific to adenocarcinomas without revealing the Pn antigen determinants of normal cells, or, respectively, the Pk antigen determinants that are associated with the determinants
K on adenocarcinoma. The percentage of false positive results is thus reduced.

 The expression "antigenic determinants K" denotes antigenic determinants which, as already stated, are specific to adenocarcinomas and which, not showing cross-reactivity with the pn antigen determinants of normal cells, are recognized only by the antibodies specific thereto, i.e. anti-K antibodies: this definition includes any CEA antigen mixture in which antigen determinants other than K determinants have been eliminated totally or substantially.

 The expression "k antigen determinants" refers to antigenic determinants that are present on adenocarcinoma cells in "association" with, ie, with the K antigen determinants that are specific for adenocarcinomas, said antigen determinants pk. showing, as already mentioned, a cross-reaction with the pn antigen determinants of normal cells: this definition includes any CEA antigen mixture in which the antigen determinants other than the determinants pk have been eliminated in whole or in quasi-all.

The pn antigen determinants are, as already mentioned, antigen determinants present in plasma or normal serum and cross-reactive with the antigen determinants pk
An anti-antibody is either an antibody obtained by immunizing animals with the above-mentioned K antigen determinants or any anti-CEA antibody (i.e., the antibody produced by immunizing animals with the modified CEA, such as so that the antibody sites other than the anti-K sites have been saturated in whole or in almost all of them with antigenic determinants specific to them.

Similarly, an anti-Pk antibody is either an antibody obtained by immunizing animals with the aforementioned pk antigen determinants or any modified ani-CEA antibody so that antibody sites other than anti pk sites have been saturated in total OR almost completely by antigen determinants which are specific
Throughout this specification, the term "antibodies" refers to both immunoglobulins and immunoglobulin fragments and may also mean the corresponding antiserum.

 Moreover, unless otherwise indicated, the term "antibody" means a polyclonal antibody; when it is a monoclonal antibody, this is specified.

 Two different methods are provided by the present invention for the preparation of anti-17 antibodies specific for K antigen determinants.

 According to the first of these methods, anti-K antibodies are obtained by absorbing antibodies produced against a mixture of antigenic determinants K and with pn or pk antigen determinants so as to saturate the immunological binding sites pk on said antibodies.

According to the second method, anti-K antibodies are 1) immunizing animals with antigenic determinants.
Pn or Pk to produce anti-Pn or anti-Pk 2 antibodies purifying a mixture of K and Pk antigen determinants
using anti-Pn or anti-Pk antibodies produced according to
1), so as to obtain R antigen determinants; and 3) immunizing animals with K antigen determinants
obtained according to 2).

 The scope of the invention includes, in addition to specific anti-K antibodies obtained by the two different methods above, also the antigenic determinants produced according to step 2) of the second method described above, and in addition, the antigenic determinants pk and and anti-Dk and anti-pn antibodies specific thereto, which are involved in the two methods above.

 The labeled forms, for example the radiolabelled forms, the enzyme-labeled forms, or the fluorescently labeled forms, of the aforementioned antigenic determinants K, pk and pn and the corresponding anti-K, anti-pk and anti-Pn antibodies are also included in the scope of the present invention.

According to the first method described above for the preparation of anti-K antibodies, the mixture of Ket antigen determinants is injected into an animal, a goat, a sheep or a rabbit, for example, following the usual and known procedures of the invention. immunization, for example in the form of an emulsion in the adjuvant of
Freund, to give birth to anti-K / Pk antiserum. The latter is recovered by taking blood from the animal according to standard techniques and then absorbed by incubation, for example, with pn or antigen determinants.
The incubation is generally between room temperature and approximately 500 ° C., preferably at about 370 ° C., for a time varying from 1 to 5 days, preferably for about three days. Normal human plasma can be used instead of the antigenic determinants Pn since, as already mentioned, normal human plasma contains pn antigen determinants. As an example, for example, 0.1 ml of goat serum can be incubated at 37 ° C. for three days with 324 ml of a solution of PBS containing pn or R antigen determinants at a concentration of approximately 50%. ml, or with about 50 ml of normal human plasma. At the end of the incubation, the substance is centrifuged and the supernatant is used as it is, or, optionally, after further dilution, purification and / or stabilization treatments, as an anti-K. specific antibody. second method described above for the preparation of antibodies or anti-K antisera, Pn or Pk antigen determinants are injected, for example in the form of an emulsion in reund's adjuvant, to an animal, to a goat a sheep or a rabbit, for example, following a standard immunization procedure, to give rise to an anti-Pn or anti-Pk antiserum, which is recovered Here again, the blood is removed from the animal in a certain manner. usual.

The immunoglobulins are extracted from the antiserum, if appropriate, by known procedures,
The purification of the mixture of antigenic determinants K and Pk by means of anti-Pn or anti-Pk antibodies to obtain the antigenic determinants K can be carried out according to known methods. One useful technique may be, by way of example, immunoaffinity or bioabsorption chromatography, which is based on the immunological reaction between an antigen and an antibody bound to an inert carrier. For example, a solution, for example a solution in PBS, of the mixture of antigens K and pk is eluted through a resin, for example Sepharose 4B, carrying anti-human antibodies. Pn or anti-Pk bound to it, thus recovering the antigenic determinants K in the unbound fraction.

 The anti-K antiserum is then produced by injecting the antigenic determinants K thus obtained into an animal, for example of the species indicated above, according to standard procedures, for example those which have already been described herein. description.

 The mixture of the K and pk antigen determinants used according to the methods described above for the preparation of anti-K antibodies is obtained by extraction of primary or metastatic adenocarcinoma tissues, for example from liver metastases, or from patients' biological fluids. with adenocarcinoma.

 The extraction can be carried out according to known procedures and, if adenocarcinoma tissues are used, they require homogenization beforehand: the homogenization is generally done with an "X Press" (LKB).

 The extraction can then be carried out using any suitable solvent, preferably a glycoprotein solvent, which may be, for example, perchloric acid, trichloroacetic acid, phosphotungstic acid and the like. , an alkali metal halide, potassium chloride for example, a mixture of phenol and ethanol, a neutral detergent, cationic or anionic such as, for example, SDS, DOC, CPC, Triton X 100 ", the" Nonidet P40 ", etc.

The extraction solvent is preferably used in volume equal to that of the homogenate or the biological fluid, and it is preferably a concentrated acid, having for example a concentration of about 0.5 N to about 2 N; Perchloric acid 2 N is an especially preferred extraction solvent. Any temperature below room temperature is suitable for the extraction process, but a temperature of about 4 ° C. is generally preferred, for example. The extraction times usually vary from about 10 minutes to about one hour.
After extraction, the precipitate is separated by filtration or centrifugation and the supernatant can be dialyzed and purified. Many known purification means can be used based on different principles.

 For example, it is possible, on the basis of differences in electrical charge, to use, for the purification of ion exchange resins such as, for example, "Sephadex" (registered trademark) DEAE, "Sephacel "(registered trademark) or -. oge -5, or "Sephadex1, QAE or SP.

On the basis of differences in molecular weight and hydrodynamic volume, chromatographic techniques, such as, for example, gel filtration and high performance liquid chromatography (HPLC) can be used.

 A higher degree of purification can be obtained on the basis of differences in electrophoretic migration and isoelectric point: thus electrophoresis techniques such as, for example, electrophoresis of inert carrier preparation can be used. for example on "Sephadex" mark pose, on gel of p. olyacrylamide, etc., or plate or column "isoelectric focusing".

 On the basis of the glycidic content, a degree can be obtained. further purification, if necessary, using different lectins attached to resins, for example "Sepharose" (Trade Mark) 4B.

 Pn antigen determinants for use in the methods described above for the production of anti-K antibodies can be obtained from normal human plasma according to standard procedures of extraction and purification.

 Thus, for example, one can extract normal human plasma with, for example, an equal volume of perchloric acid or with any other appropriate solvent such as, for example, one of those that have been indicated above for the extraction of the K / Pk antigen mixture from adenocarcinoma tissues; similar temperatures and extraction times can also be used. The extraction product can then be dialysed in a conventional manner and purified by known techniques, for example imuno-affinity with antibodies specific to for example by immunoaffinity chromatography against anti-K / Pk antibodies or anti-Pk antibodies or against anti-Pn antibodies. The pn antigen determinants thus bind to the antibodies and are detached therefrom by known methods, for example using a thiocyanate, urea or propionic acid, according to standard procedures [J.W. Eveleigh, D.E.

Levy, J. of Solid Phase Biochemistry, 2, 45, 1977].

The antigenic determinants pk which, if appropriate, can also be involved in the above methods for the preparation of anti-K antibodies can be obtained, for example, from the mixture of antigen determinants.
This mixture, for example extracted from adenocarcinoma tissues as described above, can be subjected to, for example, a bioabsorption chromatography, for example by eluting a solution of these mixtures, for example a solution in PBS, through a resin, for example "Sepharose" 4B, carrying anti-Pn antibodies attached thereto.

 The antigenic determinants pk, as a result of their cross-reaction with the antigenic determinants Pn, bind to the resin which carries the anti-pn antibodies, and are detached therefrom in a conventional manner, for example by means of thiocyanate or urea according to conventional procedures, as reported above.

The content or degree of purification of the antigenic substance, i.e., antigenic determinants K, Pk k and P n and antigenic K / P k mixtures is controlled, either by radioimmunoassay or immunochemically. from their immunological activity K and pk or pn, according to conventional methods. Thus, for example, a radioimmunoassay of the K-determinant content of an antigenic substance can be performed by measuring the inhibition produced by said substance with respect to the binding capacity of a substance. RIA system 1 gI-K # anti-K. A similar control of a 125I-Pk # anti RIA system
Pk or, respectively, of an anti-Pn 125I-Pn # RIA system makes it possible to evaluate the content of Pk or, respectively, in Pn of an antigenic substance.

Anti-P n antibodies and antibodies were. pk are obtained by known immunization techniques, for example by the same as those described above to obtain antiserum and anti-K / P antibodies k
According to the invention, monoclonal anti-K, anti-Pk and anti Pn antibodies can be produced against a single antigenic determinant K, Pk or Pn by following the known procedures which are generally used to prepare monoclonal antibodies, for example the technique known from the hybridoma as reported, for example, by
Kohler C. and Milstein C. in Nature 256, 495-497, 1975.

According to the invention, any known method for the determination of an antigen, for example any immunoassay method, for example the radioimmunoassay (RIA), or the enzymoimmunoassay (EIA) or immunofluorescence or immunoluminescence, or immunodiffusion or immunoelectrophoresis, may be used to reveal or quantitate the antigenic determinants K which are specific for adenocarcinomas or, where appropriate, the antigenic determinants Pk which are associated to determinants K on adenocarcinomas
The use of one or more of the antigenic determinants K, Pk and K / Pk in admixture, whether in their unmixed form or in their labeled form, and corresponding labeled or unlabeled antibodies, in any one of of said methods known above, makes it possible to establish an early diagnosis of human tumoral pathologies which are known under the name of adenocarcinomas
An antigen, or an antigenic determinant or an antigenic mixture or a labeled antibody is, respectively, an antigen or an antigenic determinant or an antigenic mixture or an antibody that has been labeled by any possible type of labeling means: it may for example, a radioisotope, an enzyme, or a fluorescent or luminescent substance.For labeling, any known and usual procedure can be followed, for example to mark the antigens.

To mark, for example, with the 125I or 131I radioisotopes one can follow the techniques of Chloramine
T or Lactoperoxidase (LPO) [Nature 194, 495, 1962 and, respectfully, Bioch. BioPh. Acta 251, 363, 19713 or variations of these techniques; or the Bolton and Hunter reagent [Biochem. J. 133, 529, 19731.

 For labeling, for example, with an enzyme, one can follow the techniques reported in FEBS Letter 95, 311, 1978 or in J. Histochem. Cytochem. 22, 1084, 1974.

 When using a radioimmunoassay (RIA) method for the diagnosis of adenocarcinoma, any known RIA technique may be used, for example, Clin. Chem. 19: 146, 19737. Thus, for example, the antigenic determinants K which are specific to adenocarcinomas QU the antigenic determinants pk which are associated with them on adenocarcinomas can, by way of example, be quantified in FIG. for example, a biological fluid or tissue extract, by competing with a known amount of labeled radioisotopes, for example antigenic determinants K or, respectively, pk or an antigenic K / pk mixture radiolabeled with iodine 125 or iodine 131 for reaction with a limited amount of anti-K or, respectively, anti-pk antibodies. The competitive reaction can be carried out in a conventional manner, for example by incubating at a temperature of about 4 ° C. to about 400 ° C., preferably at room temperature, for a period of time ranging from approximately 6 to 90 hours. preferably for about 15 to 16 hours.

 The antigenic fraction attached to the antibodies is then separated from the non-fixed fraction and the radioactivity is measured in the fixed and / or non-fixed fraction.

The separation of the bound antigenic fraction from the unfixed fraction can be by known methods, for example by using immunoglobulin-specific antibodies of the species in which the first antibodies were given, ie that is to say to the anti-K or anti-PK antibodies. The radioactivity in the fixed fraction and in the non-fixed fraction can be evaluated by means of a standard curve, drawn for example with a standard K or a standard pK, or by directly measuring the possible fall in the binding capacity of the radiolabeled K or Pk or K / Pk with the anti-K or anti-Pk antibodies, and thus, for example, a decrease in radioactivity measured on the fixed fraction is proportional to the concentration of the sample in K or pk
When using an enzymoimmunological assay method for the diagnosis of adenocarcinomas, any known immunological method using enzyme labeling may be used, for example, in the methods described in J. Immunclogy 109, 129, 1972. While many enzymes can be used successfully as parrers, particularly preferred enzymes are, for example, peroxidase,? -galactosidase, alkaline phosphatase and glucose oxidase.

For example, according to a possible method of enzymo-immunological assay, the antigenic determinants K or pk of a sample of biological fluids or tissue extracts can be quantified by a sandwich-type EIA procedure according to wherein the sample is reacted by incubation, for example, with anti-K or respectively anti-Pk antibodies which are either chemically bound or passively absorbed on a solid support, for example on polystyrene beads. After removing the excess reagent by washes, the enzyme-labeled anti-K or, respectively, anti-P antibodies are added, such as, for example, the ss-galactosidase labeled antibodies, obtained according to the procedure described. in
FEBS Letters 95, 311-313, 1978. After further washings, the appropriate enzymatic substrate, for example ortho-nitrophenyl-galactopyranoside, is added if s-galactosidase is used as the label, and measures the color developed, its intensity being proportional to the concentration of the antigenic determinants R or, respectively, pk in the sample. For histochemical application, any known immunohistochemical method can be used [Human Pathology 12, 590-596, 1981] to identify the presence of K or Pk antigenic determinants on histological preparations, using specific anti-human antibodies. K or anti-Pk, according to the invention. The histological determination can be done on tissue sections frozen or fixed and routinely wrapped, according to conventional procedures.

 Thus, for example, tissue sections can be incubated at room temperature in an aqueous buffer with anti-K or anti-Pk antibodies, so that the anti-K antibodies or anti-pk Pk bind to sites where the respective antigenic determinants K or pk are present. The fixed antibodies are then detected by subsequent reaction, for example by incubation with labeled antibodies specific for immunoglobulins of the species in which the first anti-K or anti-Pk antibodies have originated.

 These labeled antibodies can be prepared according to known procedures using known markers such as, for example, enzymes, for example peroxidase, β-galactosidase, alkaline phosphatase, glucoseoxidase, enzyme-anti-enzyme complexes, for example the peroxidase-antiperoxidase complex (PAP); fluorescent molecules, for example fluorescein or Xodamine or IBiotinmAvidiX combinations in which the "Avidin" is itself attached to an enzyme or a fluorescent substance, for example of the type mentioned above.

 The final microscopic analysis of the histological preparation will reveal the sites where the desired K or pk antigenic determinants are present: fluorescent sites will be observed when a fluorescent marker is used, while colored sites will be observed when used for the determination of a marker enzyme and a specific chromogenic substrate.

 A particularly useful combination for the histochemical detection of K or pk antigenic determinants on tissue sections involves the enzyme β-galactosidase as a label and an indolyl-galactoside, e.g. 5-bromo-4. Chloro-indolyl-galactoside, as enzymatic chromogenic substrate: for example, the immunogalactosidase technique described in, for example, Histochemistry 76, 153-158, can be followed for such a determination. , 1982.

But, as already mentioned, in addition to the immunoassay methods described above, any other immunological method according to the invention can be used to demonstrate the presence of antigenic determinants K specific to adenocarcinoma or associated antigenic Pk determinants in biological fluids or in extracts or tissue sections
As has already been said, in addition to the methods for diagnosing human adenocarcinomas, the invention also provides combinations of reagents for carrying out said methods.

 Although different combinations of reagents are provided for different analytical methods, a combination of reagents for the detection of antigenic determinants K still contains, as an essential component, an anti-K specific antibody, and a combination of reagents allowing the detection of the antigenic determinants Pk contains, as an essential constituent, an anti-Pk antibody. Each of said reagent combinations contains, in addition, other constituents which are possibly different from one combination of reagents to the other, depending on the analytic method to which the combination of reagents is intended, and these additional components may be, for example, selected from labeled K or Pk antigenic determinants, labeled K / Pk antigenic mixtures, labeled anti-K or anti-Pk antibodies, or labeled antibodies specific to immunoglobulins of the species in which it said anti-K or anti-Pk antibodies have originated.

 In addition, other constituents, optional or not, may optionally be found in the combinations of reagents that are the subject of the invention, such as, for example, a separation system without binding, controls, standards, buffers, stabilizers, antimicrobial agents, surfactants, enzyme substrates and activators.

Thus, for example, for a radioimmunoassay method for detecting antigenic determinants K or pk in biological fluids or in tissue extracts according to the method.
RIA described above in this specification, a usable reagent combination may consist of a "kit" containing essentially 1) an anti-K or, respectively, anti-Pk antibody; 2) the respective antigenic determinants K or Pk radio
labeled, or an antigenic mixture K / Pk radio
marked 3) witnesses; and 4) buffers.

 The above kit may further contain a second immunoglobulin-specific antibody of the species in which the respective anti-K or anti-Pk antibody of component 1) has been developed, and, optionally, surfactants and or stabilizing or antimicrobial agents.

 The radiolabeled K or Pk or K / Pk of component 2) of the kit may be, for example, K or Pk or K / Pk labeled with 125I or 1311 radioisotope.

 The controls 3) comprise at least one negative control and one positive control, and the buffers 4) can comprise any buffer having, for example, a pH of between about 6.5 and about 8, preferably equal to 7, About 2.

 The surface-active substances may be, for example, "Tween" 20 or 80, "Nonidet P40," Triton X100 ", etc.

 Stabilizers may be, for example, beef serum albumin, gelatin, etc.

 An antimicrobial agent may be, for example, sodium azide (NaN3).

The buffers may be, for example, phosphate buffers, optionally mixed with inorganic salts such as, for example, sodium chloride
In a most preferred embodiment, a kit for RIA detection of antigenic determinants K or Pk contains: 1) an anti-K or anti-Pk antibody from a goat,
properly diluted in a mixture of:
- phosphate buffer whose pH is between 0.5
about eL 8, preferably about 7.2,
whose molar concentration is between
About 20 mmoles and about 130 mmoles, preferably
equal to about 70 mmolec
- sodium chloride whose molar concentration is
between about 20 mmol and about 150 mmol
preferably equal to about 70 mmol,
- EDTA sodium salt, the molar concentration of which is
between about 25 mmol and about 50 mmol,
preferably equal to about 33 mmoles
- sodium azide whose molar concentration is
between about 15 mmol and about 50 mmol,
preferably equal to about 31 mmol, and
- normal goat serum whose volume concentration /
volume is between about 0.0% and 0.5%
preferably about 0.1%. 2) A reference buffer (to be used to determine the
maximum binding capacity of the antibody system ++
radiolabeled antigen) comprising
a phosphate buffer whose pH is between
About 6.5 and about 8, preferably 7.2
approximately, and whose molar concentration is included
between about 20 mmoles and about 150 mmoles,
preferably equal to about 70 mmoles
sodium chloride whose molar concentration is
between about 20 mmol and about 150 mmol,
preferably equal to about 70 mmoles
- sodium azide whose molar concentration
is between about 15 mmol and 50 mmol
about, preferably about 31 mmol; and
- serum albumin of beef whose concentration
weight / volume is between about 0.1% and 1%
about 0.4%, preferably about 3). A marker comprising:
antigenic determinants K or Pk or a mixture
antigenic K / Pk radiolabeled with iodine 125, diluted in
reference buffer 2) so as to have a concentra
1.5 ng / ml with a specific activity of approximately
60 mCi / mg; 4) a goat anti-immunoglobulin antiserum precipitating,
correctly diluted in reference buffer 2) 5) negative control: negative human plasma tested,
that is, normal human plasma; and 6) a positive control: test positive human plasma,
that is, human plasma from a patient with
of adenocarcinoma or normal human plasma to which a
known quantity of antigenic determinants K or Pk or
a K / P antigenic mixture was added.

For an enzymo-immunological assay method for detecting K or pk antigenic determinants in biological fluids or tissue extracts according to the sandwich-type EIA method described hereinabove, a combination of reagents containing essentially, for example 1) anti-K or antipk antibodies attached to
a solid phase; 2) the respective anti-K or anti-Pk antibodies conjugated to
an enzyme 3) an enzymatic substrate; 4) witnesses; and 5) buffers.

 The combination of reagents above may further contain other components such as, for example, a blocking agent for the enzymatic reaction and, in addition, as already known, surface-active substances, stabilizers, antimicrobial agents, enzymatic activators, etc.

 The anti-K or anti-P antibodies of component a) can, for example, be absorbed on polystyrene beads.

 Component 2) conjugated to an enzyme may be, for example, anti-K or anti-Pk antibodies conjugated with the enzyme β-galactosidase, according to known methods already set forth in this specification.

 Component 3), an enzyme substrate, may be, for example, any chromogenic substrate which is specific for the enzyme of component 2). If X-galactosidase is used as the enzyme, an appropriate chromogenic substrate can be used. for example, ortho-nitrophenylgalactopyranoside.

 Controls 4) and buffers 5) may be those that have already been specified for the RIA kit.

The possible blocking agent of the enzymatic reaction may be, for example, when it is S-galactosidase which is used as an enzyme, an alkali metal carbonate, sodium carbonate for example. In a particularly preferred embodiment, a kit for detection by the aforementioned sandwich-type EIA method of K or Pk antigenic determinants comprises 1) anti-K or anti-Pk antibodies absorbed
on polystyrene beads 2) a diluent buffer containing
a phosphate buffer whose pH is between
About 6.5 and about 8, preferably 7.2
approximately, and whose molar concentration is included
between about 50 mmol and about 150 mmol of
preferably equal to about 70 mmoles
- sodium chloride whose molar concentration
is between about 50 mmol and 150 mmol
about, preferably about 70 mmoles
- sodium azide whose molar concentration
is between about 15 mmol and 50 mmol
about, preferably about 31 mmol;
and
- serum albumin of beef whose concentration
weight is between about 0.5% and 3%
about, preferably about 1%; 3) the respective anti-K or anti-pk antibodies conjugated to
-galactosidase, properly diluted in the buffer
2); 4) an enzymatic substrate containing
ortho-nitrophenylgalactopyranoside, the
molar concentration is between 2 mmol
about 4 mmol, preferably equal to
About 2.3 mmoles; and
a phosphate buffer whose pH is between
About 6.5 and about 8, preferably 7.2
approximately, and whose molar concentration is
between about 10 mmol and about 30 mmol,
preferably equal to about 15 mmol; 5) an enzymatic reaction blocking agent, which
contains sodium carbonate at a concentration
molar range from about 0.25M to about 2M,
preferably equal to 1 M about 6) a negative control: negative human plasma under test,
that is, normal human plasma; and 7) a positive control: positive human plasma test,
that is, human plasma from a patient with
adenocarcinoma, or normal human plasma that has been
added a known amount of antigenic determinants
K or pk or antigenic mixture K / pk
For the histochemical assay of K or pk antigenic determinants on tissue sections according to the procedure already described herein, a combination of reagents that may be used may be, for example, a kit containing essentially 1) anti-K antibodies or respectively anti-Pk 2) enzyme-labeled antibodies that are specific to
immunoglobulins of the species in which the antibodies
respective anti-K or anti-Pk of component 1) have originated 3) an enzymatic substrate; and 4) buffers.

Other components may be included in the above kit such as, for example, a blocking agent for removing potentially troublesome nonspecific interactions, a contrast dye (for the nuclei), and, in addition, have already said, surfactants, stabilizers, antimicrobial agents, enzyme activators, etc.
In the combination of reagents above, one can give rise, for example, anti-K or antipk antibodies in the goat, then the antibodies of component 2) will be anti-goat immunoglobulin antibodies and will be grown, for example, at the donkey. for example, the antibodies labeled with the enzyme of component 2) may be antibodies labeled with β-galactosidase, obtained by known techniques as has already been said.

 The enzymatic substrate component 3) can be any substance which is specific for the enzyme of component 2) and which is capable of giving a product which is coorinated and insoluble by the action of the enzyme: if it is 6-galacto- sidase which is used as an enzyme, a useful chromogenic substrate may be, for example, an indolylgalactoside, in particular 5-bromo-4-chloroindolylgalactoside.

 The buffers may be, for example, phosphate or "Tris" buffers having a pH of from about 6.5 to about 8 and is preferably about 7.3.

 The blocking agent that may be required to eliminate interferences resulting from nonspecific interactions may be, for example, normal donkey serum for the case where the antibodies of component 2) are born in the ne.

 The contrast dye may contain, for example, carminic acid.

According to a particularly preferred embodiment, a kit allowing the histochemical demonstration of the antigenic determinants K or pk on tissue sections contains 1) a washing buffer comprising
a buffer "Tris" or phosphate whose pH is included
between about 6.5 and about 8 and is preferably
equal to about 7.3, and whose molar concentration
is between 5 mmol and 100 mmol and
is preferably equal to about 10 mmol; and
- sodium chloride whose molar concentration
is between about 50 mmol and 200 mmol
about and is preferably equal to 150 mmol
about 2) a blocking buffer comprising the same constituents
washing buffer 1) above and, in addition, the
normal donkey serum at a volume / volume concentration
between about 1% and about 5% and
preferably about 3% 3) anti-K or anti-respective antibodies grown in
the goat, properly diluted in the buffer 1) ci
above ; 4) Conjugated goat anti-immunoglobulin antibodies
with 3-galactosidase, grown in the donkey and correctly
diluted in buffer 1) above 5) an enzymatic substrate containing
a phosphate buffer whose pH is between 7
approximately and approximately 8 and is preferably equal to 7.4
approximately, and whose molar concentration is included
between about 5 mmol and about 50 mmol and is
preferably equal to about 10 mmoles
- sodium chloride whose molar concentration
is between about 5 mmol and 50 mmol
about and is preferably about 10 mmol
magnesium chloride whose molar concentration
is between about 0.1 mmol and 2 mmol
about and is preferably equal to about 1 mmol
5-bromo-4-chloroindolylgalactoside, the
molar concentration is between 0.5 mmol
about 5 irmoles and is preferably
equal to about 1 mmol
- potassium ferrocyanide whose concentration
molar is between about 3 mmol and 10
mmoles and is preferably 6 mmol
about ; and
- potassium ferricyanide, the concentration of which
molar is between about 3 mmol and 10
mmoles and is preferably equal to 6 ntmcles
about ; 6) a contrast coloring agent containing
carminic acid whose weight concentration
volume is between 1 gXl and about 10 g / l and
and is preferably about 2.5 g / l; and
- potassium sulfate and dealuminium whose
molar concentration is between 25 mmol
about 150 mmol and is preferably
equal to about 50 mmol.

 In the preferred embodiments described above for the combinations of reagents according to the invention, the concentrations indicated are the concentrations per liter to which each constituent is present in the solution or in the mixture used to carry out the test.

 The use of the reagent combinations which are the subject of the present invention in known analytical methods for the determination of antigens, including liquid-phase immunoassay methods and solid-phase immunoassay methods and histochemical determinations significantly improves the ability to diagnose human adenocarcinoma.

 The possibility of detecting, according to the invention, only the antigenic determinants (K) which are specific for adenocarcinomas without interference resulting from the presence of the pn antigenic determinants or the antigenic determinants (P k) which are associated with the K determinants on adenocarcinomas , makes it possible to achieve better diagnoses than those obtained with the known commercially available kits for the CEA assay, with the methods and reagents according to the invention, there is a markedly reduced number of false positive results.

 The results obtained from experiments carried out with the methods and reagents according to the invention on numerous cases of non-neoplastic pathologies, including numerous pathologies of inflammatory degeneration, and also of neoplastic pathologies, have been characterized by percentages. False positive results and, respectively, false negative results, lower than those observed in cases where the known CEA kits are used, using anti-CEA antibodies (eg Roche CEA test and CEA kit) of Liso-phase).

Especially when comparative experiments have been carried out by analysis of histological preparations with the reagents according to the present invention, on the one hand, and with commercially available kits, on the other hand, for example, DAKO PAP. KIT "and 'IIMMULOR HISTO
SET ", it has been observed that while the known kits give a positive reaction even on certain normal cells, with a particularly high percentage of error, for example in the case of marrow tissue analysis, the The reagents according to the invention exclusively reveal adenocarcinoma metastatic cells, without any false positive result, that is to say without any positive staining of normal cells, granulocytes for example, which is not at all the case. The new anti-K, polyclonal or monoclonal antibodies, or the anti-Pk antibodies, the subject of the present invention, as well as the anti-human antibody fragments, were compared with the commercially available kits to which they were compared. K or anti-Pk, for example fragments
Fab 'or F (ab') 2 can be used as markers, for example, in nuclear medicine and radioimmunoassay. For example, it is possible to label said antibodies or antibody fragments with radioisotopes, for example, I31I, and to inject the radiolabel thus obtained into the human body in order to visualize and locate the possible cells or sdenocarcinoma masses. The display of the marker can be done by known methods with the aid of special instruments, for example by means of a gamma camera.

Another application of new anti-K, polyclonal or monoclonal antibodies, or new anti-antibodies
Pk, or fragments thereof, which are the subject of the present invention is the therapy of human adenocarcinomas The possibility of using specific anti-K or anti-pk antibodies that are capable of recognizing cells exclusively. adenocarcinoma without recognizing normal cells, allows for tumor chemotherapy that is specifically and exclusively directed to tumor masses.

 For this application, said novel antibodies or antibody fragments can be conjugated with antitumor drugs or other cytotoxic agents, and the resulting product can be injected into the patient so that the drug or cytotoxic agent is not specifically transported. by the antibody carrier only to the cells or tumor masses.

 Antitumoral drugs may be, for example, daunorubicin, doxorubicin, epirubicin and methotrexate.

 A cytotoxic agent may be, for example, ricin.

 The conjugation of the antitumor drug or the cytotoxic agent, for example one of those mentioned above, with a polyclonal or monoclonal anti-K or anti-pK antibody according to the invention can be carried out according to known procedures, for example those described in J. Immun. Methods 59, 129-143 (1983). The conjugates formed between an antitumor drug or a cytotoxic agent, for example one of those already indicated, and a polyclonal or monoclonal anti-K or anti-Pk antibody, constitute another subject of the invention.

 The present invention therefore also provides an improved method of specific and focused chemotherapy of human adenocarcinomas, wherein the effect of the drug is developed exclusively on the tumor cells and normal cells are not affected by the drug. .

In the present specification, the abbreviations EDTA, "Tris", PBS, PEG, HPLC, IgG, mCi, mM,
P / V, V / V, DEAE, SDS, DOC, CPC are respectively ethylenediamine tetraacetic acid, tris (hydroxymethyl) aminomethane, phosphate buffered saline, polyethylene glycol, high performance liquid chromatography , one or more immunoglobulins, the milli
Curie, number of millimoles, weight / volume ratio, volume / volume ratio, diethylaminoethyl cellulose, sodium dodecyl sulfate, sodium deoxycholate, and cetyl pyridinium chloride
The following examples illustrate the invention without however limiting it in any way.

EXAMPLE 1
100 g of hepatic adenocarcinoma liver metastases were frozen at -80 ° C for 24 hours and then homogenized under high pressure using an LKB X-press instrument. with three volumes of a 0.025 M aqueous solution of sucrose, then an equal volume of 2N HCl04 was added thereto. After centrifugation (10,000 g for 20 minutes at 40 ° C.) and dialysis of the supernatant with distilled water, precipitated with 3M KCl for 24 hours at 40 ° C. with stirring The mixture was again centrifuged (100,000 g for one hour at 40 ° C.) and the supernatant was dialyzed with 0.005 M phosphate buffer at pH 6 75.

The RIA extract was checked from the inhibition produced on the binding capacity of 125I-K # anti-K or 125I-K / Pk # anti-K system and then chromatographed. in a DEAE cellulose column (1.6x5 cm) previously equilibrated with 0.005 X phosphate buffer at pH 6.75. Successive elutions were carried out with 40 ml of a 0.025 M phosphate buffer at pH 6.75, with 40 m of a 0.05 M phosphate buffer at pH 6.75 and then with 40 ml of a 0.1 M phosphate buffer at pH 6.75. Each fraction eluted by RIA was checked as above, then concentrated to 5 ml. The 0.025 M and 0.05 M fractions were further purified by gel filtration (4 ml / hr) in a column of "Sephadex" G 200 (~ 1.6 cm, height 100 cm) buffered to pH. 4.65 with an aqueous solution containing 0.05 M NaH2PO4 and 0.9% NaCl. Two peaks were thus eluted and each of them was again controlled by RIA, as indicated above, and also with a 125I-Pk # anti-Pk system in order to know its antigenic activity content K and Pk. Both peaks were confirmed to be a mixture of antigenic determinants K and pk with a concentration of
K greater in the first peak than in the second.

EXAMPLE 2
5 liters of normal human plasma were centrifuged at 5,000 g for 15 minutes at 4 ° C. and the supernatant was then extracted with an equal volume of 1.2M HClO4.

The extract was dialyzed with running water for 24 hours and then again with bidistilled water for another 24 hours. After concentrating 10 to 20 times with an "Amicon" cell and dialysis with
PBS at pH 7.2, was added to the 0.5% "Tween" 80 extract (to avoid nonspecific interactions), and then chromatographed in a 2 "bicsorption column. Sepharose ™ (Trade Mark) 4B carrying the anti-Pn antibodies bound to the resin.

 The fraction bound to the resin during the elution process (Pn antigenic determinants), then detached from the resin itself using a solution of ammonium thiocyanate in 3M PBS, according to the procedure described in J. of Solid-Phase Biochemistry 2, 45, 1977.

The product obtained was dialyzed with distilled water and PBS pH 7.2, and then monitored by
RIA by measuring the inhibition of the attachment capacity of an anti-Pn 125I-Pn # system.

 It was thus confirmed that the content corresponded to antigenic determinants p n at a concentration of 50 / ml determined by the Lowry method.

EXAMPLE 3
A solution of PBS at pH 7.2, containing Tween 80 (0.5%) - and the K / Pk antigenic mixture (1 mg / ml) obtained in Example 1 (first peak of elution), a chromatography in a column of bioabsorption of "Sepharose" 4 B fixing antipk antibodies.

 The unbound fraction containing the antigenic determinants K was concentrated to a small volume and was monitored by RIA by measuring the inhibition of the binding capacity of an 125 I-K anti-K RIA system.

 It was thus confirmed that the unbound fraction contained antigenic determinants K at a concentration of 0.1 mg / ml in the mixture of PBS and Tween.

 The bound fraction, i.e. the fraction bound to the anti-Pk antibodies from the column, was detached by elution with ammonium thiocyanate solution in 3M PBS, according to the procedure. as in Example 2. The eluate was then dialyzed with bidistilled water and then with PBS, and its contents were controlled by RIA from the inhibition of the attachment capacity of a 125I RIA system. -Pk # anti-Pk. It was thus confirmed that the eluted fraction contained antigenic determinants pk at a concentration of 0.15 mg / ml.

The same procedure was repeated above using a "Sepharose" bioabsorption column 4B binding the anti-P antibodies. During the elution of the antigenic mixture k, the antigenic determinants pk are crosslinked with the anti-Pn antibodies of the column, and this has been proved by the fact that antigenic determinants P k, at a concentration of 0, 1 5 mg / ml, were recovered from the fixed fraction.The Pk content control was again performed by RIA, as indicated above using a 125I-Pk # anti RIA system.
pk
EXAMPLE 4
A solution in PBS of the K / P antigenic mixture obtained according to Example 1 was emulsified at a concentration of 1 mg / ml, with an equal volume of a complete suspension of adjuvant εd. The resulting mixture was used to make four subcutaneous injections in four different places in the back of a Tibetan goat. The injections were repeated three times at fifteen-day intervals. After the third injection, the immunological response was checked: blood was taken from the animal and the serum titer was checked as follows. incubate serial dilutions of the serum overnight at 37 ° C with 100 L of a PBS solution containing 1.5 ng / ml 125 I radiolabeled antigenic determinants K or, alternatively, a K / Pk antigenic mixture radiolabeled with iodine 125.

after incubation, 0.1 ml of a solution was added
1/10 goat anti-immunoglobulin PBS in rabbits and 0.1 ml of 1/8000 PBS solution of normal goat serum. The mixture was then centrifuged at 5000 g for 15 minutes at 4 ° C., the supernatant was removed, and the radioactivity was measured with a gamma counter on the precipitate. The titre of the antiserum was calculated as an antibody dilution capable of binding 50% of the total labeled antigen. It has been found that K / Pk antiserum
obtained had an average title of 1 / 50,000.

 By following a similar procedure but using, in place of the antigenic mixture K / Pk, antigenic determinants K or antigenic determinants pk obtained according to Example 3, or antigenic determinants Pn obtained according to Example 2, we have obtained respectively anti-K antiserum, antipk antiserum and anti-P antiserum.

EXAMPLE 5
1 ml of the anti-K / Pk antiserum obtained in Example 4 was treated with a 14% PEG 6.000 solution in 9 ml of a 0.1M Veronal buffer at pH 8.6. .

 The resulting precipitate was taken up with the smallest possible amount of 0.02 M phosphate buffer having a pH of 8, and purified on DEAE cellulose.

 The eluted proteins were again precipitated in the starting buffer (5 ml) with a solution of PEG 6,000 at 20% in a 0.1 M Veronal buffer at pH 8.6 (45 ml).

The precipitate was taken up with PBS and 1 ml of 0.5% Tween 80 and then purified again in a Sephadex bioabsorption column 4B carrying attached K / Pk antigens.

 Anti-K / Pk immunoglobulins which remained attached to the column were then removed by elution with ammonium thiocyanate according to the procedure set forth in Example 2 to obtain anti-K / Pk antibodies.

 By a similar procedure, starting from anti-K, anti-Pk and anti-Pn antisera obtained in Example k 4, anti-K, anti-k and anti-Pn antibodies were obtained respectively.

EXAMPLE 6
The anti-K / Pk antiserum obtained according to the procedure of Example 4 (0.1 ml) was incubated for 24 hours at 370 ° C. with 8 ml of normal human plasma, and the mixture was centrifuged at room temperature. 5,000 g for 5 minutes at 40C. Each 0.1 ml portion of the supernatant was re-incubated for 24 hours at 370 ° C. with 4 ml of normal human plasma, and again centrifuged under the same conditions as indicated above.

 The supernatant was separated and its immunological activity was monitored from its ability to bind to the same extent 125 I radiolabelled K / Pk antigenic mixture, both in the presence and on the other hand. Absence of Normal Human Plasma The absence of cross-reactivity with normal human plasma, i.e. with Pn antigenic determinants, proved that the antiserum obtained was anti-K antiserum.

The serum obtained was diluted appropriately and used for immunological determinations of antigenic determinants K on human plasma or other biological fluids or tissue extracts.
EXAMPLE 7
370C was incubated for 24 hours with anti-K / Pk antiserum obtained according to the procedure of Example 4 (0.1 ml), with a solution in PBS at 50 μl / ml of antigenic determinants Pn obtained by the procedure of Example 2 (1.8 ml). After incubation, the mixture was centrifuged at 5,000 g for 15 minutes at 4 ° C.
Each 0.1 ml portion of the supernatant was again incubated for 24 hours at 370 ° C. with the PBS solution at 50 μg / ml of Pn antigenic determinants (1.8 ml). After centrifugation under the same conditions as above, the supernatant was taken to appropriate dilutions and used as anti-K antiserum for immunohistochemical determinations.

The anti-K immunological activity of the antiserum was checked from its ability to reveal adenocarcinoma cells but not normal cells, on histological sections: for this test, the staining method was followed by peroxidase, as described by Sternberger LA in Immunocytochemistry - 2 Edition
John Wiley and Sons - New York, 1979.

EXAMPLE 8
BALB / c mice, 8 weeks old, were immunized by intraperitoneal injection of the emulsion obtained by mixing antigenic determinants K prepared according to the procedure of Example 3 (100 g) with an equal volume of adjuvant complete Freund
After 30 days, intraperitoneal injections were repeated, with lesser amounts of K antigenic determinants. Immunological response was monitored on the animals, as shown in Example 4.

 Then using the control positive animals, somatic hybridization was performed with murine myeloma cells according to the procedure described in Nature 256, 495-497, 1975.

 The specificity of the products obtained by RIA test was evaluated by measuring the binding capacity of a 125I-K antigen.

 Hybrids that tested positive were then isolated and cloned.

Large amounts of anti-K monoclonal antibody were obtained by intraperitoneal injection into mice
BALB / c cells secreting monoclonal anti-K antibody.

 The growth of the tumor due to the injected cells was manifested by the production of an ascitic fluid containing up to 20 mg / ml of monoclonal anti-K antibodies.

By operating in an analogous manner but using for the immunization of the antigenic determinants Pk or Pn, instead of the antigenic determinants K, dn obtained respectively monoclonal antibodies anti-Pk and monoclonal antibodies antip
EXAMPLE 9
A solution of antigenic determinants K prepared according to the procedure of Example 3 (10 μg) in 20 μl of a solution of PBS at pH 7.2 was treated for 5 minutes in an ice bath with Na125I solution (1 mCi) in 10 μl of PBS, and 20 μl of a solution containing chloramine T in PBS at a concentration of 0.5 mg / ml.

 20 l of a solution containing sodium metabisulfite in PBS at a concentration of 2 mg / ml, and 20 μl of a solution containing potassium iodide in PBS at a concentration of 20 mg / ml. The reaction product was purified by HPLC to remove free iodine 125, and 125 I-radiolabeled K (125I-K) antigenic determinants were quantitatively obtained.

 By a similar procedure, radiolabelled Pk antigenic determinants (125 I-Pk) and radiolabeled K / Pk antigenic mixtures (125 I-K / Pk) were prepared.

EXAMPLE 10
Using anti-K antiserum obtained in Example 6, samples of human serum from 335 hospitalized patients with non-neoplastic diseases were tested using the reagents and the analytical method indicated below.

Reagents (1) Reference pad containing
70 mmol of phosphate buffer at pH 7.2;
70 mmol of sodium chloride,
31 mmol of sodium azide; and
albumin serum of beef at 0.4 t P / V.

(2) Anti-K antiserum obtained as in Example 6, correctly
diluted to 50/1000 in
70 mmol of phosphate buffer at pH 7.2;
70 mmol of sodium chloride;
33 mmol disodium salt of EDTA;
31 mmol of sodium azide; and
- normal goat serum at 0.1% V / V.

(3) Radio-iodinated marker containing an antigenic mixture
125I-K / Pk diluted in the buffer (1) so as to have a
concentration of 1 5 ng / ml and a specific activity
60 mCi / mg.

(4) Second antiserum: antiserum precipitating immune
Goat globulins, having originated in rabbits
and properly diluted in the buffer (1).

(5) Negative control of normal human plasma.

(6) Positive control: normal human plasma auguel was
added a known amount of antigenic mixture K / Pk
Analytical method
"Zero" Witness Witness Echan
positive negative tillon
Reference buffer (1) 0.5 ml - -
Negative control (5) - 0.5 ml -
Positive control (6) - - 0.5 ml
Sample - - - 0.5 ml
Antiserum anti-K (2) 0.2 ml 0.2 ml 0.2 ml 0.2 ml
Mix, incubate for 5 hours at room temperature
Radio-iodine marker (3) 0.1 ml 0.1 ml 0.1 ml 0.1 ml
Incubate overnight at room temperature
Second antiserum (4) 0.2 ml 0.2 ml 0.2 ml 0.2 ml
Incubate for 1 hour at room temperature, centrifuge for 30 minutes at 4000 rpm, decant and measure the radioactivity in the precipitate using a gamma counter.

Calculation: the PI positivity index was calculated by evaluating the radioactivity of the samples and, respectively, of the controls, relative to the radioactivity of the zero "according to the following expressions sample activity
PI (sample) = 100 - x 100
activity of "zero"
PI (control) = 100 - x 100
activity of "zero"
A PI value greater than 15 was considered to indicate the presence of adenocarcinoma, while a PI value of less than 15 was considered to indicate the absence of adenocarcinoma.

 The above 335 samples were also examined in parallel using two commercially available kits for the detection of human CEA, namely the Roche CEA assay, and the Liso-phase CEA kit from Lepetit.

The results obtained are summarized in the following table:

<tb> False <SEP> positive <SEP> results
<tb><SEP> Number <SEP> Pathologies <SEP><SEP> Test of <SEP><SEP> Test <SEP> Our
<tb><SEP> of <SEP> cases <SEP> Roche <SEP> Lepetit <SEP> test
<tb><SEP> 30 <SEP> Cirrhosis <SEP> of <SEP> liver <SEP> 4 <SEP> (13.2 <SEP>%) <SEP> 3 <SEP> (19 <SEP>%) <SEP> 2 <SEP> (6.6 <SEP>%)
<tb><SEP> 17 <SEP> Diseases <SEP> inflamma- <SEP> 4 <SEP> (23.5 <SEP>%) <SEP> 5 <SEP> (23.5 <SEP>%) <SEP > 2 <SEP> (11.7 <SEP>%)
<tb><SEP> lungs <SEP>
<tb><SEP> 18 <SEP> Broncho-pneumopa- <SEP> 3 <SEP> (16.6 <SEP>%) <SEP> 3 <SEP> (16.6 <SEP>%) <SEP> 1 <SEP> (5.5 <SEP>%)
<tb><SEP> obstructive thie <SEP>
<tb><SEP> 270 <SEP> Other <SEP> diseases <SEP> no <SEP> 4 <SEP> (1.5 <SEP>%) <SEP> 4 <SEP> (1.8 <SEP>% ) <SEP> 1 <SEP> (0.3 <SEP>%)
<tb><SEP> neoplastics
<tb><SEP> 335 <SEP> Total <SEP> 15 <SEP> (4.5 <SEP>%) <SEP> 15 <SEP> (4.5 <SEP>%) <SEP> 6 <SEP> (1.8 <SEP>%)
<Tb>
EXAMPLE 11
Human serum samples from 84 identified adenocarcinoma patients were analyzed using the reagents and analytical method described in Example 10, and using in parallel the two commercial kits also referred to Example 10

The results are summarized in the following table.

<Tb>

<SEP> False <SEP> negative <SEP> results
<tb> Number <SEP> Pathologies <SEP><SEP> Trial of <SEP><SEP> Trial of <SEP> Our
<tb> of <SEP> cases <SEP> Roche <SEP> Lapetit <SEP> test
<tb><SEP> 60 <SEP> Adenocarcinomas <SEP> 5 <SEP> (8.3 <SEP>%) <SEP> 7 <SEP> (11.6 <SEP>%) <SEP> 2 <SEP> (3.3 <SEP>%)
<tb><SEP> Gastrointestinal <SEP> intestinal <SEP> i <SEP>
<tb><SEP> 24 <SEP> Adenocarcinomas <SEP> 2 <SEP> (8.3 <SEP>%) <SEP> 2 <SEP> (8.3 <SEP>%) <SEP> 0 <SEP> (0 <SEP>%)
<tb><SEP> of the <SEP> lung
<tb> 84 <SEP> Total <SEP> 7 <SEP> (8.3 <SEP>%) <SEP> 9 <SEP> (10.7 <SEP>%) <SEP> 2 <SEP> (2, 4 <SEP>%)
<Tb>
EXAMPLE 12
The same human plasma samples were also examined as in Examples 10 and 11, using the reagents and the other analytical method which are shown below.

Reagents (1) Anti-K antibodies obtained according to the procedure described
in Examples 5 and 6, absorbed on poly beads
Styrene (2) Diluent Buffers Containing
70 mmoles of phosphate buffer at pH 7.2
70 mmol of sodium chloride;
31 mmol of sodium azide; and
- 1% bovine serum albumin (W / V) (3) Enzymatic conjugate containing the same antibodies
anti-K than those of reagent (1) above, conjugated to
2-galactosidase (according to the procedure described
in FEBS Letters 95, 311, 1978) and properly diluted
in the buffer (2).

(4) Enzymatic substrate containing
15 mmol of phosphate buffer at pH 7.0; and
2.3 mmoles of ortho-nitrophenylgalactoside.

(5) Blocking agent: 1M sodium carbonate

(6) Negative control: normal human plasma.

(7) Positive control: normal human plasma to which
added a known quantity of antigenic mixtures K / P k
Analytical method
"Zero" Witness Witness Sample
negative positive
Diluent buffer (2) 0.2 ml 0.1 ml 0.1 ml 0.1 ml
Negative control (6) - 0.1 ml -
Positive control (7) - - O, i ml Sampling - - - 0,1 ml
Anti-K antibodies (1) 1 pearl 1 pearl 1 pearl 1 pearl
Incubate by shaking for 5 hours at room temperature.

Discard the liquid phase.

Wash twice with 2 ml of saline
Enzyme Conjugate (3) 0.2 ml 0.2 ml 0.2 ml 0.2 ml
Incubate by shaking for 2 hours at room temperature.

Discard the liquid phase.

Wash twice with 2 ml of saline
Enzymatic Substrate (4) 1 ml 1 ml 1 ml I ml
Incubate for one hour at 37 ° C.

Blocking agent (5) 1 ml 1 ml 1 ml 1 ml
Measure the absorbance at 420 nm using a spectrophotometer.

Calculation
The index of positivity PI was calculated with respect to the optical density (OD) of "zero" according to the expression:
OD of the sample
PI =
OD of "zero"
A value of the positivity index greater than 3 was considered an indication of the presence of adenocarcinoma, while a value of the positiovity index of less than 3 was considered as an indication of absence of adenocarcinoma.

 Similar results were observed to those reported in Examples 10 and 11.

EXAMPLE 13
Formalin-fixed, paraffin-embedded tissue sections from human colon adenocarcinoma, human mammalian adenocarcinoma, and bone marrow from patients with adenocarcinoma of the prostate were examined. chest, using the reagents and analytical distrust that are shown below:
Reagents (1) Wash Buffer Containing:
10 mmol of a phosphate buffer at pH 7.3 1 and
150 mmol of sodium chloride.

(2) Blocking buffer containing
10 mmol of a phosphate buffer at pH 7.3
150 mmol of sodium chloride; and
- normal donkey serum at 3% (V / V).

(3) Enzymatic conjugate containing anti-antibodies
Goat immunoglobulins grown in donkeys, marked
with 8-galactosidase (according to the method described
in FEBS Letters 95, 311, 1978, and properly diluted
in the buffer (1).

(4) Enzymatic substrate containing
10 mmol of phosphate buffer at pH 7.4;
10 mmol of sodium chloride;
1 mmol of magnesium chloride;
1 mmol of 5-bromo-4-chloroindolyl galactoside;
- 6 mmoles of potassium ferrocyanide; and
- 6 mmoles of potassium ferricyanide.

(5) Contrast coloring agent containing
- potassium bisulfate and aluminum; and
carminic acid.

(6) Anti-K antibodies raised in goats, obtained according to
the procedure of Example 7, and properly diluted
in the buffer (1) above.

Analytical method
The sections were deparaffinized in xylol, in the series of ethyl alcohols from 100% to 50%, and then in the washing buffer (1). The optional reagent was added in such a quantity that the entire cup was covered.

Blocking buffer (2).

Incubate for 20 minutes at 370C in a humid chamber.

Remove excess reagent by shaking.

Add the anti-K (6) antibodies.

Incubate for 2 hours at 370C in a humid chamber.

Add the enzymatic conjugate (3).

Incubate for 30 minutes at 370C in a humid chamber.

Wash the cup with the pad (1) for 5 minutes.

Add the enzymatic substrate (4).

Incubate for 30 minutes at 370C.

Remove excess reagent by washing.

Add the contrast dye (5)
After one minute, wash the cup under running water and observe under a light microscope.

 In another method, the above analytical procedure was followed by incubating overnight at room temperature in a humid chamber, except for incubation with the enzymatic substrate (4), which was carried out for a period of one hour. hour at room temperature in a humid chamber.

 Still as an alternative to the above procedure, after the final washing with running water and before the microscope examination, the sections were first clarified by settling them in the washing buffer (1), in the 50% to 100% ethyl alcohol series, and in xylol, and then mounted in an "Eukitt" (registered trademark).

 Microscopic analysis of the sections revealed an intense blue coloration exclusively localized to the cytoplasm and membrane of the tumor cells, without any positive staining reaction in cells of normal surrounding tissues, such as granulocytes for example.

These results proved to be very different from those obtained by analyzing the same preparations using commercially available kits for the histochemical detection of the CEA complex via anti-CEA antibodies, namely the "DAKO PAP KIT". "and the" IMMUNOLOK
HISTOSET KIT ".

 These commercial reagents have, in fact, shown much less specificity, in that they have been found to stain not only tumor cells but also, to a varying degree, normal tissue cells. This error is particularly important in the case of bone marrow tissue analysis.

 The same procedure as above was followed for the analysis of frozen tissue sections and similar results were obtained.

EXAMPLE 14
Doxorubicin was conjugated to anti-K antibodies by a covalent binding method.

 A solution of doxorubicin (40 mg / ml) in 0.015 M phosphate buffered saline pH 7.2 was mixed with a slight molar excess of 0.1 M Nais4 and incubated for one hour. hour at room temperature in the dark.

 At the end of this incubation, 1M glycerol was added to a final concentration of 0.05M.

 The oxidized doxorubicin solution was mixed with 1 ml of anti-K antibody solution (20 mg / ml) in 0.15 M potassium carbonate buffer pH 9.5, and incubate for one hour at room temperature.

At the end of the incubation, NaBH4 was added to a final concentration of 0.3 mg / ml and the reaction allowed to proceed for two hours at 370 ° C.
At the end of the reaction, the mixture was subjected to chromatography in a gel filtration column (15 x 1.5 cm) of "Bio-gel" P 100, equilibrated in PBS.

 Fractions of the excluded volume contained drug bound to the non-conjugated drug-free antibodies.

Claims (19)

 A suitable method for use in the diagnosis of human adenocarcinoma by determining antigenic material related to adenocarcinoma in a sample taken from a patient, characterized in that by using (a) an anti-human antibody Whether the monoclonal or polyclonal K or (b) an anti-onoclonal or plolyclonal antibody, the material which is determined in the sample has (a ') exclusively those of the antigenic determinants K specific for the adenocarcinomas or (b') those of the antigenic determinants Pk associated with K determinants on adenocarcinomas.  2. Antibodies selected from specific anti-K antibodies for antigenic determinants K, specific anti-Pk antibodies for antigenic determinants Pk and anti-Pn specific antibodies for antigenic determinants Pn.  A process for the preparation of the anti-K antibodies according to claim 2, said method comprising a) absorbing the antibodies produced against a mixture of antigenic determinants K and Pk with the antigenic determinants pn or pk in order to saturating the immunological binding sites k on said antibodies or (b, 1) to immunize animals with the antigenic determinants Pn or Pk to produce anti-body anti Pn or anti-Pk; 2) purifying a mixture of antigenic determinants K and pk using anti-Pn or anti-pk antibodies produced according to) thereby obtaining antigenic determinants K and 3) immunizing animals with antigenic determinants K obtained according to 2).  4. Process for the preparation of anti-Pk antibodies according to claim 2, said method of immunizing animals with Pk antigenic determinants.  A process for the preparation of anti-Pn antibodies according to claim 2, said method comprising immunizing animals with antigenic determinants Pn.  6. Antigenic determinants selected from antigenic determinants K specific for human adenocarcinomas, antigenic determinants Pk, said antigenic determinants pk being the antigenic determinants associated with antigenic determinants K on human adenocarcinomas, and antigenic determinants pn, said antigenic determinants pn being the antigenic determinants present in plasma or normal serum and cross-reacting with the antigenic determinants pk.  A process for the preparation of the antigenic determinants K according to claim 6, said method comprising purifying a mixture of antigenic determinants K and pk by means of anti-Pn or anti-Pk antibodies.  8. Process for the preparation of the antigenic determinants Pk according to claim 6, said process consisting in purifying a mixture of antigenic determinants K and Pk by means of anti-Pn antibodies.  9. Process for the preparation of the antigenic determinants pP according to claim 6, said process comprising extraction from plasma or normal serum and purification by immunoaffinity with anti-bodies specific to them.  10. Monoclonal antibodies selected from anti-monoclonal antibodies specific for antigenic determinants alone, anti-monoclonal antibodies specific for pk antigenic determinants alone and anti-monoclonal antibodies specific for determinants antigenic pn alone.  11. Process for the preparation of monoclonal antibodies according to claim 10, characterized in that said monoclonal antibodies are obtained by the hybridoma technique.  A reagent system for use in a method for the diagnosis of human adenocarcinoma according to claim 1, comprising as essential constituent anti-K monoclonal or polyclonal antibodies specific for antigenic determinants K or anti-Pk antibodies. monoclonals or polyclonals specific for antigenic determinants Pk.  A reagent system for use in a radioimmunoassay method for the diagnosis of human adenocarcinoma according to claim 1, comprising essentially 1) anti-K or anti-PK antibody 2) antigenic determinants K or respectively; antigenic determinants; as an alternative antigenic mixture labeled with a radioactive product, preferably 125 I-K or 125 I-Pk or 125 I-K / Pk 3 ) witnesses and 4) buffers.  The reagent system of claim 13, further comprising a second antibody specific for immunoglobulins of the species in which the anti-K or anti-Pk antibody of component 1) has been developed.  15. A reagent system for use in an immunoassay method with enzymes for the diagnosis of human adenocarcinoma according to claim 7, containing essentially 1) anti-K or anti-Pk antibodies fixed on a solid phase 2) anti-K or anti-Pk antibodies conjugated to an enzyme, preferably to β-galactosidase; 3) an enzymatic substrate, preferably orthonitrophenylgalactopyranoside; ) buffers.  16. A reagent system for use in an immunohistochemical method for the diagnosis of human adenocarcinoma according to claim 11, containing essentially 1) anti-K antibodies or, respectively, anti-Pk 2) enzyme-labeled antibodies. specific for the immunoglobulins of species in which the anti-K or anti-Pk antibodies of component 1) have been developed, the enzymatic label being preferably β-galactosidase 3) an enzyme carrier, preferably -bromo-4-chloroindolyl-galactoside 4) buffers.  17. K, pk and pn labeled antigenic determinants preferably labeled with an isotope or labeled with polyclonal or anti-K, anti-P and anti-P labeled monoclonal or anti-body.  44  18. Anti-K or anti-Pk polyclonal or monoclonal antibodies for use as excipients for antitumor drugs or cytotoxic agents in the therapy of human adenocarcinomas.  19. Conjugates between a polyclonal or monoclonal anti-K or anti-P A antibody, and an anti-tumor drug or a cytotoxic agent, preferably daunorubicin, doxorubicin, epirubicin, methotrexate or ricin.