NO791643L - CANCER-ASSOCIATED PROTEIN AND PROCEDURE FOR MAKING IT - Google Patents

Description

The present invention relates to the production of a

purified cancer-associated protein.

US patent 4,132,769 (corresponding to German official publication

P 25 48 636.6) describes a cancer-associated protein which

is denoted Cl IAC. Cl IAC is described as a cancer-associated glycoprotein which exhibits Cl inactivator action (Cl esterase-inhibiting action), and which has a molecular weight of 110,000-130,000 Dalton, determined by gel filtration, and a Swedberg constant of 6.2S determined by ultracentrifugation. Cl IAC was found in pleural and ascites fluids from cancer patients, serum from cancer patients and other body fluids from cancer patients,

as well as in culture media - from cultured human cancer cells.

According to the present invention, it has been found that a purified protein showing the same immunological determinants as Cl IAC (in other words, is able to precipitate

with the antibodies described in US patent 4,132,769 which are

directed specifically against Cl IAC, and capable of immunizing specifically immunizable non-human animals such as Danish landrace pigs and sheep for the formation of antibodies specific for Cl IAC as described in US Patent 4,132,769), can be obtained by passing a Cl IAC-containing solution through an antibody-affinity chromatography column containing matrix-immobilized antibodies directed specifically against Cl IAC, and eluting the column with a detergent.

The purified protein eluted in this way

(hereinafter referred to as CaDet), can be characterized in that it is precipitated with immunoglobulins that are directed specifically against the cancer-associated Cl inactivator Cl IAC which is defined in the above-mentioned US patent 4,132,769, and in that in a

SDS PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)

gradient gel essentially shows only one sharp band at approx. 25,000 Daltons.

The fact that the protein shows essentially only one sharp band in an SDS PAGE gradient gel (corresponding to 95-99%, usually at least 98% of the total protein present) is considered, in accordance with normal biochemical principles, as evidence for the establishment of a purified protein, and the specific "molecular weight" found in SDS PAGA is considered an unequivocal, characteristic "fingerprint" of the protein in question.

The purified protein (hereinafter referred to as CaDet) according to the invention, with immunological determinants which are only reactive with antibodies against Cl IAC, which purified protein in other words has immunological determinants which are characteristic of Cl IAC, is very useful as an antigen for immunization of specifically immunizable animals to have specific antibodies directed against Cl IAC (and thus also directed against CaDet) formed. The advantages of using the new protein CaDet in this production of antibodies directed against Cl IAC is in particular that due to the use of the purified antigen during the immunization, a much higher titer of the monospecific antibody is achieved (specifically against Cl IAC and thus also against CaDet ).

The hitherto unknown protein CaDet thus represents a significant advance in the production of antibodies specific for Cl IAC. The more specific and more sensitive antibodies obtained by the use of CaDet for the immunization of specifically immunizable animals entail significant advantages in the diagnostic use of anti Cl IAC. The higher specificity and sensitivity (avidity) that the anti Cl IAC immunoglobulins produced using CaDet as an immunizing agent show, enables the detection of lower concentrations of Cl IAC in the serum of cancer patients.

In addition to being useful as an antigen for the immunization of animals, CaDet can also be used for the standardization of anti Cl IAC tests, and as a reagent in hemagglutination tests and in radioimmunoassays.

Antibodies raised against CaDet, e.g. in sheep or pigs as described in the above-mentioned US patent 4,132,769, can be prepared for diagnostic use in the same way as described in this US patent. Thus, the antibodies can e.g.

is incorporated into an agarose gel where holes have been punched for the application of material suspected of being Cl IAC-containing (Manzini technique). It can also e.g. RIA tests are performed using either isotopically labeled CaDet for absorption of antibody, or isotopically labeled antibody for absorption of CaDet in accordance with common RIA principles. In addition, test methods that use the henagglutination technique can be used,

according to which CaDet is applied to red blood cells (or latex or similar particles) and the antibody is applied.

It is well known that it should be preferred to perform hemagglutination tests and analogous reactions with pure antigen or mono-specific antibody.

Another application possibility for antibodies against the purified CaDet according to the invention is for administration to humans for the therapy of various cancer diseases. For this purpose, the antibodies are produced in pigs, e.g. in pigs of the Danish landrace, and the antiserum obtained from the immunized pigs is subjected to serum fractionation to obtain the essentially pure IgG immunoglobulin fraction thereof,

as it has been shown (cf. US patent 4,132,769) that pig IgG immunoglobulins can be tolerated by humans, as they do not immunize humans by parenteral administration.

A further application for antibodies raised against CaDet is as antibodies by antibody-affinity chromatography for the preparation of CaDet from Cl IAC-containing liquids.

As mentioned above, CaDet can be prepared from a Cl IAC containing solution by antibody affinity chromatography technique and elution with a detergent. The molecular weight of

about. 25,000 Daltons according to SDS PAGE differs significantly from the molecular weight of 110,000-130,000 Daltons, determined by gel filtration, which is stated in US patent 4,132,769. However, the fact that the protein, when purified, emits a "fingerprint" in an SDS PAGE acrylamide gradient gel at a specific molecular weight does not necessarily mean that the protein in its original form has the same molecular weight.

The molecular weight of approx. The 25,000 Daltons found in the SDS PAGE gradient, compared to the molecular weight of 110,000-130,000 Daltons, previously found by gel filtration for the protein Cl IAC, may indicate that CaDet is a breakdown product

of the original cancer-associated Cl inactivator (Cl IAC),

as this degradation product e.g. is formed by the detergent-1 treatments in the elution step and/or the SDS treatment,

but has retained Cl IAC's immunological determinants,

or it may indicate that the Cl IAC described in US patent 4,132,769 is not a pure protein, but instead is a conglomerate of several proteins that have a Cl esterase-inhibiting effect.

A gel filtration of crude Cl IAC on Sephacryl S-200

in 1% Triton X-100, phosphate buffered saline (PBS) (pH = 7.2), IM NaCl resulted in the elution of three peaks which precipitate with

anti Cl IAC as described in US patent 4,132,769, with one peak corresponding to a molecular weight of 90,000-100,000 Dalton,

the second peak responded to 50,000-55,000 Dalton and the third peak responded to 25,000-30,000 Dalton. This gel filtration is described in more detail in the following experimental section.

The molecular weight found by this gel filtration appears

to be consistent with the assumption that Cl IAC is a conglomerate of several entities.

Whatever the exact nature of the protein chemical relationship between Cl IAC and CaDet, however, as explained above, CaDet has proved to be a particularly useful antigen for the production of monospecific, high-titer antibodies against Cl IAC, and these antibodies have the same application areas such as the anti Cl IAC described in US patent 4,132,769,

but they exhibit additional advantages due to their improved titer and highly monospecific character.

The invention also relates to a method for the production of a protein (CaDet) which is precipitated with antibodies specific for Cl IAC (as described in US patent 4.132.769),

and which exhibits the above pattern in SDS PAGE acrylamide gradient gel with 95-99%, usually at least 98% of the protein as a single band at about 25,000 Daltons. This method involves subjecting a solution containing a component that is precipitated with antibodies directed specifically to Cl IAC to antibody affinity chromatography using matrix-immobilized antibodies directed directly to Cl IAC, and the antibody-bound CaDet is eluted. This antibody affinity chromatography is conveniently carried out using a column of the matrix-immobilized antibodies in a manner known per se. The presence of CaDet in the eluate can be detected using the above-

said SDS' PAGE gradient gel electrophoresis on the eluate in accordance with e.g. the method described in the following experimental part, and the monitoring of the elution of CaDet is suitably carried out by spectrophotometry for detection

of the presence of protein in the eluate. The Cl IAC activity in the eluate fractions is conveniently determined by immunoprecipitation tests, e.g. by measuring the diameter of the precipitation ring in a Manzini test using anti Cl IAC as described in the above-mentioned US Patent 4,132,769.

Suitable conditions for charging the column are established

in accordance with well-known antibody affinity chromatography principles, and an example of suitable conditions appears in the subsequent experimental section. The conditions for elution of the column are adapted to the elution of the above characterized protein CaDet, and it has been shown that a suitable elution buffer is a buffer containing 0.1% Triton X-100,

IM NaCl and PBS pH value 7.2. There is no reason to believe

that Triton X-100 is the only detergent that is able to elute Cl IAC from the column without destroying the antigen (or destroying the antibody column), and it is within the scope of the invention to use other detergents of either an ionic nature such as SDS, or not -ionic species for elution of CaDet from the column.

The solution applied to the column and containing a component that is precipitated with antibodies against Cl IAC can be either a crude Cl IAC-containing preparation such as pleural or ascites fluid from cancer patients or culture medium from culture

of human cancer cells, both as described in the above-mentioned US patent 4,132,769, or it may conveniently be a partially purified preparation thereof, e.g. the preparation that has been purified by any of the adsorption and gel filtration methods described in the aforementioned US patent 4,132,769, preferably a product produced by the "Sephadex" A50 method described in US patent 4,132,769, which method involves adsorbing the Cl IAC-containing material to an anion exchanger

(e.g. "Sephadex" A50) and precipitate Cl IAC-containing material from the eluate of the anion exchanger, dialyze the precipitate, dissolve it in a NaCl solution and lyophilize. Other Cl IAC-containing starting materials suitable for use in the preparation of CaDet are placental fluid and placenta-associated fluids. A high concentration of Cl IAC has been found in the placenta, and then the placenta

is a source of human proteins available without legal or ethical restrictions, the production of antibodies against Cl IAC using placenta derived Cl IAC as antigen, preferably CaDet derived from placenta, constitutes an important aspect of the present invention.

The fact that the placenta contains Cl IAC must be taken into account

in the interpretation of diagnostic testing of samples from pregnant women using anti Cl IAC produced against Cl IAC or CaDet, but does not contradict the previously described character

of Cl IAC as a protein that is associated with several types of cancer, where Cl IAC protects the cancer cells that grow as parasites in human tissue from being destroyed by the human immune system.

In exactly the same way, Cl IAC may be the immune system-neutralizing protein that enables the fetus to attach and grow in the womb without being attacked by the mother's immune system.

Like all other biological mixtures from which a

protein that is present in small or relatively small amounts,

is to be isolated, the Cl IAC-containing starting solutions used for the production of CaDet as described here should preferably be treated to remove, neutralize or destroy proteolytic activity in them, e.g. by adding an enzyme destructor or enzyme inhibitor such as soy trypsin inhibitor, poly-L-lysine or "Trasyloi", or by passing the solution through columns of immobilized enzyme inhibitors or enzyme destructors, and the anti Cl IAC immunoglobulins used in the antibody activity column, can also, before being immobilized or used, be treated to remove any proteolytic activity therefrom by treatment in the same way, preferably by passage through a column of matrix-immobilized enzyme inhibitor or enzyme destructor.

The anti Cl IAC immunoglobulins used in the antibody affinity chromatography column can be immunoglobulins produced in a manner known per se, against Cl IAC as described in the above-mentioned US patent 4,132,769, and which preferably,

if necessary, are absorbed to increase their specific specificity, or they may be immunoglobulins raised against CaDet. The immobilization of the immunoglobulins on a matrix is conveniently carried out by binding the immunoglobulins covalently to a matrix, e.g. cross-linked agarose, such as "Sepharose" 4B,

in accordance with the well-known principles. The antibodies are preferably bound to such an extent that the total amount of antibody that is bound covalently to the matrix corresponds to a maximum of 85% of the immunoglobulins used in the covalent binding. Analogous to what is described in Scand. J. Immunol.

(5, 77-86 (1977) , this leads to the highest recovery from the column.

For storage (preferably at -70°C or at +4°C) and later use, the eluate containing CaDet is suitably concentrated to a small volume with a concentration of protein of the order of 10 times per ml or above. This concentration can e.g. carried out using dialysis tubing and sucrose or using a concentration cell (e.g. Mi II i po re) with a filter with a molecular weight cut-off of

10,000 Daltons. The presence of a detergent in the eluate stabilizes CaDet, which otherwise, like any other pure protein in aqueous solution, tends to be unstable. Other suitable and useful stabilizers are SDS (e.g. 0.1 or 0.2% solution), BSA (bovine serum albumin) (e.g. 1% solution) or other suitable non-human proteins or peptides which does not interfere with the intended use of CaDet. Alternatively, the addition of approx. 25% by volume ethylene glycol.

For use as an antigen for immunization of animals, CaDet, which is preferably stabilized with e.g. Triton X-100 or SDS, suitably combined with a carrier after it has been dialysed against e.g. 20 mmol PB, 0.1% Triton X-100. The carrier can e.g. be Blue Dextran 2000 (dextran with a molecular weight of approx. 2,000,000, to which Cibacron F3 GA is covalently bound) and similar macromolecular materials, to which a ligand is attached that binds proteins in accordance with the same mechanism as Cibacron F3 GA. It is also useful to combine it with Freund's adjuvant (complete or incomplete). For use in immunization, CaDet can also be bound to various other carriers, which in themselves are known for immunization purposes, e.g. PBD or albumin from the animal species being immunized.

The immunization of specifically immunizable animals, e.g. pigs, is conveniently carried out in the same way as described in the above-mentioned US patent 4,132,769, and as an example, a pig can be immunized by 4 injections in the neck with an interval of approx. 14 days, with as little as 2 gamma CaDet in aqueous solution stabilized with e.g. 0.1% Triton X-100, combined with an equal volume of Freund's adjuvant. The monitoring of antiserum and the collection of antiserum from the animal can be carried out in accordance with the methods described in US patent 4,132,769, preferably using CaDet as antigen for monitoring the antibody titer.

Although antibody affinity chromatography using a column has been described above, it is clear that any other antibody affinity chromatography method using immobilized antibody may be used for the purposes of the present invention.

The treatment can e.g. is carried out in portions, or the chromatography can be carried out in centrifuges in a manner known per se, and these obviously equivalent possibilities are within the scope of the present invention.

As an alternative to antibody affinity chromatography, other affinity chromatography mechanisms can be used in a similar way, as in all cases a material is used as matrix-immobilized material to which Cl IAC and CaDet have an affinity, e.g. enzymes that are inhibited by Cl IAC,

particularly proteases such as plasmin and activated Cl-esterase, and other materials to which Cl IAC has an affinity, e.g. lectins such as Con A, hydrophobic ligands, Blue Dextrin and other materials containing a ligand of the same type as Cibacron F3 GA.

Experimental part

Gel filtration

For further characterization of raw Cl IAC by determining a molecular weight or several molecular weights thereof in a mild detergent and comparing this with the molecular weight of CaDet, a gel filtration is carried out in "Sephacryl G-200"

(a gel prepared by covalently cross-linking allyl-dextran with N,N'-methylene-bis-acrylamide, exclusion range 5000-200,000 Dalton). The gel buffer is IM NaCl, 20 mmol phosphate buffer (PB), pH value 7.2, containing 1% Triton X-100.

The molecular weights determined in this way must be assumed to be approximately similar to the molecular weights of the original forms of crude Cl IAC.

Experiments are carried out in the following way:

"Sephacryl G-200" is packed in a column Pharmacia.K 2.6/100 (diameter 2.6 cm, length 100 cm) using 2-6 layer volumes of gel buffer. After a further 3 layer volumes of gel buffer, the column is stabilized. The column is then calibrated using 5 known molecular markers, with molecular weights of 12,400, 17,800, 25,000, 45,000 and 67,000 respectively, the markers being dissolved in 5 ml of gel buffer. After the application of the molecular markers, the column is washed with gel buffer. Fractions of 5.3 ml are collected,

and the corresponding values for optical density at 230 nm are measured using a flow-through cuvette to equilibrate the column. One vial of lyophilized, semi-purified Cl IAC (prepared as described in the above US Patent 4,132,769, column 31) is dissolved in 5 ml of gel buffer. After application of this sample, fractions of the eluate are collected, and measurements of optical density at 230 nm are carried out, as described above. This leads to a protein elution curve as shown in fig. 3. To remove excess Triton X-100, fractions 16-60 are individually "dialyzed" using small columns of G 10 Sephadex (agarose gel), which are pre-calibrated in 0.1% Triton X-100 +

20 mmol PB ("dialysis buffer") in the following way:

First, the individual fractions are applied to the G-10 columns, then washing is carried out with approx. 2-3.5 ml "dialysis buffer", depending on the volume of applied material. The eluted ("dialyzed") samples are tested directly by a Manzini anti Cl IAC test (cf. US Patent 4,132,769), each dialyzed sample responding

until the gel filtration fraction is applied in a punched hole in the Manzini plate. After 4 to 8 hours, the diameter of the precipitation ring is determined. The results are listed graphically in fig. 3 ..

As can be seen from fig. 3 there is a small peak at 90,000-100,000 Dalton, and a distinct (largest) peak at 50,000-55,000 Dalton, followed by a shoulder at 25,000-30,000 Dalton. A comparison of these results with CaDet's

SDS PAGE pattern justifies the assumption that CaDet (as

is identical or nearly identical to pure Cl IAC) exhibits a complex molecular weight or complex molecular weights in nonionic detergent. The Cl IAC complexes may have been formed from the 25,000 Dalton sample, as both samples corresponding to 25,000, 50,000 and 100,000 Daltons can be observed in the above-mentioned gel

filtering. When the same gel filtration is carried out without detergent, a broad peak in the range 75,000-125,000 Dalton is obtained.

Preparation of CaDet.

10 ml anti Cl IAC (absorbed) produced in pigs

and treated and absorbed as described in US Patent 4,132,769, mixed with 10 ml of NaHCO 3 (0.1M). containing 0.5M NaCl,

pH value 8.3. 2 g of dry CNBR-activated "Sepharose 4B" gel

-3

(cross-linked agarose) is suspended and washed in 10 M HCl

(400 ml in total). After filtration (Buchner funnel), the gel is mixed with the above-mentioned antibody solution at room temperature and under slow stirring for 2 hours. After the coupling with antibody, the gel is washed with "loading buffer" (0.1M sodium acetate, pH 7.2) containing 1% ethanolamine. The gel is degassed in vacuum and packed into a chromatographic column, Pharmacia K9/10 and washed with 10 layer volumes of loading buffer and then with 10 layer volumes of elution buffer (IM NaCl + 0.1% Triton X-100, PBS pH value 7.2) . This wash cycle is carried out 5 times. The column is then ready for application of material containing proteins that can react with antibody against Cl IAC. In this case, the same lyophilized Cl IAC-containing material is used as was used for the gel filtration described above. The lyophilized material is dissolved in 5 ml of the loading buffer described above. The column is washed with loading buffer, until the protein that is not bound to the column is washed out. All fractions washed from the column and recorded on a flow-through cuvette spectrophotometer are termed "washing liquid". The column is then eluted using the above-mentioned elution buffer in fractions of 5 ml. The eluate is poured together and concentrated using dialysis tubing/sucrose to a volume of approx. 2 ml. This is termed "eluate". The same concentration operation from 40 ml to approx. 2 ml is carried out with the above "washing liquid". The crude Cl IAC-containing protein applied to the column is termed "loaded material". The loading operation, washing and elution are carried out at 4°C.

"Introduced material", "washing liquid" and "eluate" are tested

in the presence of protein that binds to anti Cl IAC described in US Patent 4,132,769. This determination is performed using cast Manzini agarose gel plates containing pig antiserum. In 5 microliter punched holes in the plate is applied

samples for "introduced material", "washing liquid" and "eluate".

In all three cases, a precipitate that looks like the precipitates in fig. 2.

The table below lists the results of the Manzini test:

The precipitation zones listed in the above table are the diameter of the precipitate measured on the basis of the Manzini technique

(SRID)

It appears from this that there is. Cl IAC activity in "input", "washing liquid" and "eluate". The same is found in another attempt (no. 471).

"Introduced material", "washing liquid" and "eluate" are subjected to SDS PAGE electrophoresis (length 15 cm, 8-20% gradient, thickness 0.75 mm, selected as a suitable method). The results of the experiments on "introduced material", "washing liquid" and "eluate" under the above conditions are shown in fig. 1. It is seen that 85-99% of the fraction of cancer-associated protein that is run under these conditions appears as one band (cf. fig. 1, no. 5 and 9, pooled eluate of cancer-associated protein fraction) . On the basis of the calculation from the molecular markers at 12,400, 17,800, 25,000

and 45,000, shown by fig. 1, no. 1, no. 6 and no. 10, it will be seen that the molecular weight of the fraction of cancer-associated protein is approx. 25,000 in this system (under non-reducing conditions, i.e. without mercapto-ethanol).

Immunization of pigs.

For immunization of pigs, 1-5 gamma CaDet is dissolved together with 0.1% Triton X-100 in 1 ml of saline solution, and the solution is mixed with 1 ml of Freund's adjuvant (complete or incomplete).

The injection into the pig and the collection of antiserum etc. are carried out as described in the above-mentioned US patent 4,132,769. After 3-5 injections with an interval of 2 weeks, the specific antibody in the pig which forms a sharp precipitate against the raw Cl IAC and against Cl IAC as described in the above-mentioned US patent is ascertained. However, as determined by the size of the precipitate rings in the Manzini technique, the specific antibody produced against CaDet has a titer of approx.

10 times as high as the titer obtained when immunizing with the Cl IAC described in the above-mentioned US Patent 4,132,769. In the same way as the antibodies raised against Cl IAC, the anti-CaDet-raised antibodies react by precipitation against the cancer-associated protein Cl IAC produced as described in the above-mentioned US patent 4,132,769, and against CaDet which is purified by the elution technique specified in the present description, as well as against sera and body fluids from cancer patients, as it e.g. the Manzini technique described in US patent 4,132,769 is used. An example of a suitable Manzini plate (at about three times magnification) is shown

in fig. 2. Sera from healthy, non-pregnant individuals (not suffering from clinically detectable cancer) show no precipitation, either against antibody produced according to US patent 4,132,769 or against antibody produced against CaDet. With the Ouchterlony technique, identity is established between anti Cl IAC antiserum as described in US patent 4,132,769 and anti CaDet.

Additional properties of the cancer-associated protein.

The lyophilized Cl IAC sample used as starting material in the above-mentioned gel filtration treatment is subjected to characterization with respect to the pH sensitivity of its antigenic genes. The following results are obtained:

pH sensitivity with regard to antigenic properties of Cl IAC

It is noted that the antigenic properties are destroyed at pH value 3.5 and below and at pH value 10.0 and above.

The sensitivity of the same Cl IAC preparation with respect to

its antigenic properties, the ability to withstand different chemicals, is investigated. The results appear in the table below:

h X) The reason for the larger ring-shaped precipitation, which indicates a greater antigenic effect, may be that these chemicals dissolve aggregates of cancer-associated proteins of the type described here.

Despite the presence of 1% ethanolamine, the antigenic properties are somewhat reduced due to the presence of 3M KSCN.

It will be seen that the antigenic properties are lost by exposure to 3MKSCN alone, and that the antigenic properties appear

to be retained in other cases. However, heating always destroys the antigenic properties.

Alternative material for immunization of animals.

According to a particular feature of the present invention, it has been found that an immunization of animals can be carried out, albeit with less outstanding results than those obtained with CaDet, using a superimposed liquid, which is prepared as described below: osteogenic sarcoma cells of human origin are grown in monolayers in Eagle's MEM with Hank's salt and 5% fetal calf serum with added glutamine as described in the above-mentioned US patent 4,132,769. The cells + the culture liquid are then frozen to -30°C, after which the resulting mixture is centrifuged, and the supernatant liquid is used to immunize pigs in the same way as described above. It is believed that the freezing leads to cytolysis or cell destruction during the release of proteins, including an antigen.

It is noteworthy that this supernatant, after mixing with Freund's adjuvant, could be used directly for the immunization of pigs. By this immunization, the pigs formed an antibody that reacted specifically with CaDet and Cl IAC, and with human cancer sera or body fluids from cancer patients.

After the cultivation and freezing of human cancer cells described above, any desired purification thereof, e.g. is carried out using the method described above for the preparation of CaDet from a crude Cl IAC-containing material.

In accordance with the principles described in US patent 4,132,769, the antibodies produced against Cl IAC can be subjected to modification or fragmentation to obtain fragments or modifications thereof in exactly the same way as described in the US patent.

Claims (15)

1. A protein, called CaDet, which precipitates with immunoglobulins directed specifically against the cancer-associated Cl inactivator (Cl IAC) defined in US patent 4,132,769, characterized in that in an SDS PAGE acrylamide gradient gel it shows essentially only one sharp band at 25,000 Dalton. 2. Protein (CaDet) according to claim 1, characterized in that it is combined with an immunogenic material. 3. Antibodies produced against the protein CaDet characterized in claim 1. 4. Antibodies according to claim 3, which are essentially pure pig IgG immunoglobulins. 5. Process for the production of a purified protein, designated CaDet, which precipitates with immunoglobulins directed specifically against the cancer-associated Cl inactivator (Cl IAC) defined in US patent 4,132,769, and which in an SDS PAGE acrylamide gradient gel shows in essentially only one band at 25,000 Dalton, characterized in that a solution containing a component that precipitates with antibodies directed specifically against Cl IAC, is subjected to affinity chromatography, and bound CaDet is eluted. 6. Method according to claim 5, characterized in that the affinity chromatography is an antibody-affinity chromatograph in . 7. Method according to claim 6, characterized in that the antibodies are matrix-immobilized antibodies directed specifically against Cl IAC. 8. Method according to claim 6, characterized in that the antibodies are matrix-immobilized antibodies produced against CaDet. 9. Method according to claim 6, characterized in that a solution containing the component which f<*> precipitates with antibodies, directed specifically against Cl IAC, are passed through an antibody-affinity chromatography column containing a matrix-immobilized antibody directed specifically against Cl IAC, after which the column is eluted to release antibody-bound CaDet. 10. Method according to claims 5, 6 or 9, characterized in that the elution of CaDet is carried out using a detergent. 11. Method according to claim 10, characterized in that the detergent is Triton X-100. 12. Method according to claim 5, characterized in that the solution which is passed through the column is prepared from a source which can be serum from cancer patients, body fluids from cancer patients, culture media from the cultivation of human cancer cells, and placental fluid or placenta-associated fluids. 13. Method for producing antisera or antibodies against Cl IAC or CaDet, characterized by immunizing a specific, immunizable non-human animal with CaDet and collecting serum from the immunized animals. 14. Process for the production of Cl IAC as described in US patent 4,132,769 or CaDet, characterized in that placental fluid or placenta-associated fluids are used as the source for raw Cl IAC. 15. Method according to claim K, characterized in that placental fluid or placenta-associated fluids are subjected to purification and concentration methods chosen from among adsorption, gel filtration and affinity chromatography.