NO832905L - CARCINOEMBRYONAL ANTIGEN, PROCEDURE FOR ITS INSULATION AND ITS USE - Google Patents

Description

With the carcinoembryonic antigen (CEA) it turns

refers to a glycoprotein with electrophoretic beta^-mobility and an apparent molecular weight of approx. 200,000 Daltons which can be detected in a small concentration of a few ng/ml in serum and/

or plasma in healthy patients, in patients with benign diseases above all in the intestine, liver and pancreas in transitory moderately elevated concentrations up to approx.

20 - 30 ng/ml or constant low elevated mirror and is typically formed in patients with a majority of malignant diseases (predominantly gastrointestinal carcinomas, pancreatic carcinoma, lung carcinoma, metastatic breast carcinoma) in the tumor and is included with clearly highly elevated concentrations (up to a maximum of over 10,000 ng/ ml) in the blood or other body

liquids. Its essential clinical importance consists in thera-

follow-up and progress control (surgery, hormone, radio and chemotherapy) of tumor patients, as a large proportion of them develop

changes in the CEA mirror and can often precede clinical

ical development (review in Klin. Wschr. 53, 193 - 203, 1975).

A plurality of methods of extraction, purification

and isolation of CEA are discussed in the scientific literature (overview see above). Most often, extraction was used

of tumor tissue using perchloric acid. The further purification takes place in most methods by combining a gel filtration step with an electrophoresis step, in some methods also by additional application of an immunoadsorption.

A CEA from is also suitable as starting material

a corresponding cell culture.

It has now been found that without an electrophoresis step, a very pure CEA is obtained from CEA-containing perchloric acid extracts when

a) high molecular weight proteins are separated by gel filtration,

b) from the eluate by immunoadsorption, the proteins with CEA antigenicity are selected, which c) are separated by liquid chromatography with high-resolution columns (hereinafter referred to as "high-performance chromatography).

The CEA thus obtained is electrophoretically heterogeneous, but immunologically uniform. It is suitable for the production of radioiodine-labeled derivatives for diagnostic purposes.

In the following, preferred embodiments according to the invention are explained in more detail: The separation of the higher molecular proteins takes place by gel filtration and suitable columns using cross-linked dextrans with a protein fractionation area which also separates proteins with a clearly higher molecular weight than for CEA. The eluate is fractionally collected, the CEA content is determined using standard analytical methods, the corresponding CEA-positive fractions are combined and concentrated.

Depending on the content of the extract of components cross-reacting with CEA (predominantly NCA = nonspecific cross-reacting antigen), a pre-purification takes place already at this stage during separation of the later eluted (because lower-molecular) NCA.

It is possible, but not necessary, to purify the eluates by means of an additional chromatography step. For this, a column with a narrower separation range of the protein fractions can be used, for example with a fractionation range from 5 10 3 to 5 10 5 D. In this way, a better separation of CEA and NCA can often be achieved.

In this way, of the higher molecular resp. also lower molecular weight proteins liberated CEA-positive fractions are divided in the next step according to the invention by means of immuno-adsorption. For this, a suitable adsorption column is charged with pre-purified IlgG globulins of a polyclonal anti-CEA antiserum which was partially recovered by immunization of rabbits or goats, or monoclonal anti-CEA antiserum. On this affinity chromatography column, the CEA-positive fractions from the previous step are present and the non-bound proteins are eluted first. Anti-body-bound proteins are then eluted with a desorption buffer and the eluates are concentrated.

It is possible to coat another affinity chromatography column with attached normal perchloric acid lung extract with a polyclonal anti-CEA antiserum as the antibodies bind to NCA and are eliminated from the antiserum.

Furthermore, it is possible to prepare an additional affinity chromatography column with coupled IgG antibodies against human serum proteins such as albumin, alpha-α-antitrypsin and acidic alpha1-glycoprotein. By chromatography of CEA-containing preparations on this column, it is possible to remove serum protein contaminants that had adsorbed non-specifically in the first affinity chromatography.

The fractions thus formed contain next

of intact CEA and NCA also CEA fragments that had likewise reacted with the immunoadsorption column's anti-CEA antibodies. For further separation it therefore joins an HPLC step.

In this last step, the CEA-positive fractions from the previous steps are separated on a high-resolution HPLC column.

You thus get the following fractions:

1. Molecular weight range 250,000 - 150,000: pure intact CEA,

II. Molecular weight range 150,000 - 80,000: immunoreactive CEA fragments (not suitable for immunization) and III. Molecular weight range 60,000 - 40,000: intact NCA and lower molecular weight CEA fragments.

After concentration, Fraction I can be used directly for a radioiodine labeling of CEA according to the chloramine-T method.

"The concentrated fractions can be desalted using

of a suitable column and then lyophilized.

The invention shall be explained in more detail by means of

some examples.

The percentages here refer to weight

if nothing else is stated.

The CEA extract used in the examples was obtained after a modification of the method of Krupey et al.

(Immunochem. 9, 617, 1972):

Pooled tumor tissue (primary tumors: Colon carcinoma

i.a., liver metastases, per extraction approx. 500 - 1000 g moist weight) is stored at -70°C, thawed for processing and coarsely crushed with scissors and a knife and then with a mechanical crusher (Braun-MULTIMIX, VIRTIS-Homogenizer "60" from

company Cenco: 30,000 rpm, 1 hour, 4°C) with approximately the same volume of distilled water and finally homogenized with an apparatus according to Potter-Elvehjern (60 ml vessel, from the company Braun) and a motorized glass pestle to a finely dispersed

porridge. This tissue slurry mixture or highly CEA-containing body fluid (e.g. pleural exudate or ascites, CEA conc. above 1000 ng/ml) is mixed with an equal volume of 1.2-molar perchloric acid and stirred for 30 minutes at room temperature. After late centrifugation (1000 g, 20 - 30 minutes, room temperature) the precipitate is discarded and the supernatant is transferred immediately if it appears clear or if it appears cloudy (fatty) only after pre-filtration ("Blauband" filter no. 589, company Schleicher&Schiill) in dialysis tubing (company Kalle, Nieder-lassung der Hoechst Aktiengesellschaft, caliber 38, length up to 100 cm) and dialyzed in this in a container with liquid tap water and continuous flow in and out (1-3 l/min.) for 24 - 48 hours. The dialysate is then mixed (approx. 1-31

with NaN^(0.02%) and evaporated using a concentration device (company Amicon, ultrafiltration cell "UF 400"

with dialysis membrane PM 10, reservoir of 4 1) to approx. 100 ml, make up to 10 times the volume with sterile double-distilled water and evaporate again to 100 ml. This final concentrate is centrifuged (10,000 g, 1 hour, 4°C) and the supernatant is lyophilized after ultrafiltration (Sartorius filter diameter 25 mm, 0, 2, 0.6 and 3.0 um). The perchloric acid extract formed from an initial quantity of 500 - 1000 g of moist tissue gives a final weight of 500 - 5000 mg.

Examples

1. Gel filtration

250 - 350 mg of extract is gel filtered over a column (2.5 x 80 cm) which is filled with N,N'-methylenebisacrylamide cross-linked allyl extrane with a bead diameter of 40 - 150 ym and a protein fraction range of 2 x 10 4 to 8 x 10 <6>"Sephacryl S 400" from Pharmacia) (column 1) using 0.05 molar tris-HCl buffer of pH 6.5 (tris-(hydroxymethyl)aminomethane hydrochloride, buffer 1). The elution rate was 20 - 25 ml/hour, individual fractions were combined to 5 ml.

The following elution diagram emerged (measurement of transmission at 280 nm):

L. Little foretop (fraction 1),

2nd intermediate zone (fraction 2),

3. broad main peak with a steep rise (fraction 3) and

4. flatter fall (fraction 4).

The collected single fractions of 5 ml were sub-

searched in the immunodiffusion test with anti-CEA reference anti-

serum or in CEA-RIA and united in the above-mentioned collective fractions.

Fraction 1 is free of CEA (and NCA), in the fractions

2-4, the amount of CEA decreases successively and the portion of NCA increases from fraction 3. \

If desired, a better separation of CEA- and NCA-containing individual fractions can be achieved by rechromatography of fractions 2-4 (individually or combined). A "Sephacryl S 200" column (2.5 x 80 cm, protein fraction range from 5 x 10 3 to 2.5 x 10 5 , column 2) serves this purpose. This gives fractions 5-7, with fraction 5 mainly containing CEA and fractions 6 and 7 mainly NCA.

Fractions 2-4 resp. 5-7 are dialyzed in a concentrator/dialysis device (evaporation vessel with collodion sleeves, company Sartorius) against a buffer (0.15-molar sodium chloride in 0.02-molar sodium phosphate buffer, pH 7.3 with 0.02% sodium azide, buffer 2) and evaporate to 0.5 - 1.5 ml.

If you use an agarose column "Sepharose 6B" (Pharmacia, column 3) instead of column 1, you get the same

led to good results.

If you use instead of column 2 "Sephadex G 200"-

column (Pharmacia company, column 4), then a comparable separation is achieved.

2. Affinity chromatography (immunadsorption)

The anti-CEA antiserum required for the immunoadsorption column was produced by immunizing rabbits with highly purified CEA or prepurified CEA-containing perchloric acid extract.

It was absorbed with lyophilized normal serum (approx. 10 mg/ml)

and adult human A/B erythrocytes (after decomplementation of

the antiserum) until non-reactivity (immunodiffusion, microscopic examination).

After the absorption of antiserum has been completed, an IgG preparation takes place by continuous anion exchange chromatography on a "DEAE-Sephacel" column (diethylaminoethyl cellulose, Pharmacia, column 5). As starting buffer, 0.01-molar sodium phosphate buffer, pH 8.0 (buffer 3) is used and as limiting buffer 0.5-molar sodium phosphate buffer pH 4.5 (buffer 4), as it is eluted with an extremely concave pH and molarity gradient ( e.g. using "Ultrograd" gradient mixer, company LKB). The selection of CEA-reactive pure anti-CEA-IgG fractions (initial fractions, fraction 8) takes place after immunodiffusion or in the immunoelectrophoresis test, temporally later eluted CEA-reactive antibodies which are, however, contaminated with other proteins are collected separately and used as antisera for the immunodiffusion, or immunoelectrophoresis (fraction 9).

To prepare the immunoadsorption column, a column (1.5 x 20 cm) is filled with 10 - 20 g of a cross-linked dextran (cyan bromide-activated "Sepharose 4 B" from the company Pharmacia, column 6) and 100 - 200 mg of the chromatographically purified IgG globulin of anti-CEA antiserum (fraction 8). On this column, 2 - 4 ml of fractions 2-4 resp. 5 - 7 and is eluted slowly (20 seconds/drops) with buffer 2 during 2-4 hours (transmission measurement at 280 nm). After complete elution of the non-bound proteins, the antibody-bound proteins are eluted with a desorption buffer (buffer 2 with 2.0 m KSCN, pH 7.3, buffer 5) (10 sec./drops). Thereby, a high sharp gradient appears in the elution diagram which, with further elution, eventually falls to a plateau which, due to KSCN transmission influence at 280 nm, lies clearly above the output level (100% transmission line). After complete elution (plateau line parallel to the 100% line), the column is regenerated with buffer 2, as in the elution diagram the plateau lines again fall to the 100% output line of the transmission measurement.

The following fractions are collected:

Fraction 10 (non-bound proteins),

fraction 11 (intermediate fraction) and

fraction 12 (bound proteins). Fraction 12 contains intact CEA, CEA fragments and NCA.

Fraction 10 is concentrated to 0.5 - 1.0 ml by dialysis against buffer 2 (collodium sleeves) and tested for CEA/NCA. Fraction 11 is discarded. Fraction 12 is dialyzed against 0.05 m sodium phosphate buffer pH 7.5 (buffer 6) and evaporated to 50 - 200 µl.

When column 6 is additionally charged with anti-CEA-IgG antibodies from an antiserum that was previously absorbed with normal spleen or lung tissue extract (highly NCA-containing) dg therefore free of antibodies against NCA, contains fraction 12

in this case no more NCA.

When, due to non-specific absorption from column 6, fraction 12 still has serum protein impurities (e.g. albumin, alpha-α-antitrypsin, acid alpha-glycoprotein), these can be removed by immunoadsorption over a further column 7 with attached IgG antigens against human serum proteins resp. perchloric acid-extracted serum proteins, since the first eluted fraction (fraction 13) contains serum protein-free

CEA/NCA.

In addition to intact CEA and NCA, the fractions used for further processing also contain CEA fragments.

3. High performance chromatography

The final isolation of CEA, NCA and CEA fragments takes place by high-resolution steric dissolution chromatography with an HPLC column ("Ultro-Pac TSK" G 4000 SW, 7.5 x 600 mm, protein fraction range 5000 - 1 million, company LKB , column 8). An HPLC device from the company LKB was used ("Multi-Perpex" pump, UV flow photometer "Uvicord S", fraction collector "Ultro-Rac II", 2-channel flat-storage printer). 50 - 500 µg protein is applied to 50 - 250 µl of the evaporated fraction 12. It is eluted with buffer 6 at an elution rate of 6 ml/hour in 350 µl fractions over a total separation time of 5 hours.

In the elution diagram (measurement of transmission and absorption with variable sensitivity from 0.01 - 0.1 at 280 nm) there are three main peaks, the middle peak having a shoulder, i.e. consisting of at least 2 sub-fractions.

Corresponding to the elution diagram, the following fractions were collected:

Fraction 14 (main fraction, CEA-reactive),

fraction 15 (CEA reactive) and

fraction 16 (NCA-reactive and CEA-reactive).

After a calibration of the HPCl column with reference proteins (thyroglobulin (molecular weight 330,000) up to alpha-lactalbumin (molecular weight 14,400)), the molecular weight of the fractions formed can be assessed as follows: Fraction 14: 250,000 - 150,000 D,

fraction 15: 150,000 - 80,000 D,

faction 16: 80,000 - 40,000 D.

After concentration to 100 - 200 µl in buffer 6, the fractions can be immediately used for a radioiodine labeling according to the chloramine-T method. They can also be desalted on a "Sephadex G 25" column (2.0 x 20 cm, column 9) and then lyophilized, obtaining final protein amounts between 100 and 1000 <µg.>

Fraction 14 contains highly purified intact CEA and fraction 16 intact NCA, while fraction 15 contains immunoreactive CEA fragments such as can also be used for immunization.

4. Iodination of CEA

20 µg CEA in 20 µl 0.05-molar sodium phosphate buffer

of pH 7.4 (the following "buffer 7") is mixed with approx. 1.5 m

125

CiNA-J (debuffered with 10 µl of 0.5-molar buffer 7) and after adding the chloramine-T solution (20 µg of chloramine T in 100 µl of 0.05-molar buffer 7) the reaction mixture is stirred for 60 seconds. The reaction is then stopped by the addition of sodium metabisulphite (20 ug Na2S2O5 in 50 ul 0.05-molar buffer 7).

It is mixed for a further 30 seconds and 0.2 ml bovine serum albumin solution (1% bovine serum albumin, 0.05-molar buffer 7, 0.1% NaN-.) is added for stabilization.

The purification of the <125>J-labelled CEA takes place by high-performance chromatography separation of the iodination mixture over a column 8.

Elution buffer: 0.0 5-molar buffer 7, 0.1% NaN3, flow: 0.1 ml/min.

Specific activity: approx. 30 m Ci/mg, immunoreactivity: approx. 90%.

Claims (10)

1. Carcinoembryonic antigen (CEA) obtainable from CEA-containing perchloric acid extracts by a) separation of high molecular weight proteins by gel filtration, b) selection of the proteins with CEA antigenicity from the eluate by immunoadsorption and -c) separation by liquid chromatography with high-resolution columns. 2. Method for isolating CEA from CEA-containing perchloric acid extracts, characterized in that a) higher molecular proteins are separated by gel filtration, b) proteins with CEA antigenicity are selected from the eluate by immunoadsorption c) are separated by liquid chromatography on high-resolution columns. 3. Method according to claim 2, characterized in that the higher molecular weight proteins are separated by gel filtration on cross-linked dextrans. 4. Method according to claim 2 or 3, characterized in that for immunoadsorption a column is charged with IgG globulins of an anti-CEA antiserum. 5. Method according to one or more of claims 2-4, characterized in that by liquid chromatography with high-resolution columns, CEA is separated as a fraction with a molecular weight range of 250,000 - 150,000. 6. Method according to one or more of claims 2-5, characterized in that the CEA-containing eluate of the liquid chromatography after concentration is used for a radioiodine labelling. 7. Method according to claim 6, characterized in that the radioiodine labeling takes place according to the chloramine-T method. 8. Radioiodine-labelled CEA, obtained according to claim 6 or 7. 9. Use of CEA according to claim 1 for the production of diagnostics. 10. Diagnostics containing a radioiodine-labeled CEA according to claim 8. 3