Carcinoembryonic antigens

Claim 2

A PROCESS FOR PRODUCING HUMAN CARCINOEMBRYONIC ANTIGEN COMPONENT A WHICH COMPRISES A. HOMOGENIZING ADENOCARCINOMA TISSUE, B. SEPARATING THE SOLID PARTICLES FROM THE HOMOGENATE, E. TREATING THE SUPERNATANT WITH A GLYCOPROTEIN SOLVENT IN WHICH THE CARCINOEMBRYONIC ANTIGEN COMPONENT A IS SOLUBLE, D. SEPARATING THE PRECIPITATE AND CLARIFYING THE RESULTING SOLUTION, E. DIALYZING THE CLARIFIED SOLUTION AGAINST A POLYETHYLENE GLYCOL WITH AN AVERAGE MOLECULAR WEIGHT OF ABOUT 15,000 TO 20,000 AND A SOFTEINING POINT AT 60*C, F. DISSOLVING THE RETENTATE IN AN AQUEOUS BUFFER OF PH FROM ABOUT 5 TO 9, G. SUBJECTING THE RESULTING MATERIAL TO SEQUENTIAL CHROMATOGRAPHY ON TWO DIFFERENT GEL COLUMNS, THE FIRST OF WHICH IS AND AGAROSE GEL HAVING ABOUT 6 PERCENT BY WEIGHT AGAROSE AND A PARTICLE SIZE OF FROM 40 TO 210 MICRONS AND THE SECOND OF WHICH IS A HYDROPHILIC WATER-INSOLUBLE CROSSLINKED DEXTRAN POLYMER GEL, H. COLLECTING AND CONCENTRATING THE ELUATES WHICH HAVE A SPECTROPHOTOMETRIC ABSORPTION PEAK WAVE LENGTH OF 280 M$ AND A MOLECULAR WEIGHT OF ABOUT 200,000 TO 500,000, I. SUBJECTING THE RESULTING ELUATES TO CHROMATOGRAPHY ON A MIXED BED CELLULOSE ION EXCHANGE COLUMN COMPRISING MICROGRANULAR DIETHYLAMINOETHYL CELLULOSE IN THE FREE BASE FROM HAVING ROD SHAPED PARTICULES, A CAPACITY OF 1.0 $0.1 MEQ./GM, A PARTICLE SIZE DISTRIBUTION OF ABOUT 20 $ TO ABOUT 60$ AND A WATER REGIN OF 2.3 TO 2.8GM/GM. DRY A MIXED BED CELLULOSE ION EXCHANGE COLUMN COMPRISING WITH A CAPACITY OF 1.0$0.1 MEQ/GM. HAVING ROD SHAPED PARTICLES, A PARTICLE SIZE DISTRIBUTION OF ABOUT 20 $ TO ABOUT 60$ AND A WATER REGIAN OF 2.3 TO 2.7 GM./GM. EXCHANGER, J. COLLECTING THE ELUATES RESULTING FROM ELUTING WITH AMMONIUM ACETATE AT PH 4 IN 0.05 M SODIUM CHLORIDE.

Claim 3

The process of claim 1 wherein prior to step (e) and subsequent to step (d) the clarified solution is dialyzed against distilled water.

Claim 4

The process of claim 1 wherein in step (g) the material is first dissolved in an aqueous buffer of about pH 7 then run through the agarose gel column which separates the CEA material, component A or component B containing fraction from higher and lower molecular weight material and colloidal particles, the eluates having a molecular weight ranging from about 200,000 -500,000 and a reading with a peak at 280 m Mu on a UV spectrophotometer are collected, dialyzed against polyethylene glycol of molecular weight about 20,000, the retentate dissolved in an aqueous buffer of pH about 7 and run through a cross-linked dextran gel column which has a higher resolving power than the first column and separates CEA material, component A or component B containing fraction from higher and lower molecular weight material.

Claim 5

The process of claim 1 wherein in step (i) the eluate is dialyzed against polyethylene glycol of molecular weight of about 20,000, the retentate dissolved in an aqueous low ionic strength buffer of pH about 4, then run through said mixed bed ion exchange column.

Claim 6

The process of claim 1 wherein in step (i) the eluate is dialyzed against polyethylene glycol of molecular weight of about 20,000, the retentate is dissolved in an aqueous ammonium acetate buffer with 0.1 M acetate of pH about 4, then run through said mixed bed ion exchange coLumn.

Claim 7

A process for producing human carcinoembryonic antigen component B which comprises a. homogenizing adenocarcinoma tissue; b. separating the solid particles from the homogenate; c. treating the supernatant with a glycoprotein solvent in which the carcinoembryonic antigen component B is soluble; d. separating the precipitate and clarifying the resulting solution; e. dialyzing the clarified solution against a polyethylene glycol with an average molecular weight of about 15,000 to 20, 000 and a softening point at 60*C.; f. dissolving the retentate in an aqueous buffer of pH from about 5 to 9; g. subjecting the resulting material to sequential chromatography on two different gel columns, the first of which is an agarose gel having about 6 percent by weight agarose and a particle size of from 40 to 210 microns and the second of which is a hydrophilic water-insoluble cross-linked dextran polymer gel; h. collecting and concentrating the eluates which have a spectrophotometric absorption peak wave length of 280 m Mu and a molecular weight of about 200,000 to 500,000; i. subjecting the resulting eluates to chromatography on a mixed bed cellulose ion exchange column comprising microgranular diethylaminoethyl cellulose in the free base form having rod shaped particles, a capacity of 1.0 + or - 0.1 meq./gm., a particle size distribution of about 20 Mu to about 60 Mu and a water regain of 2.3 to 2.8 gm./gm. dry exchanger and a microgranular carboxymethycellulose with a capacity of 1.0 + or - 0.1 meq./gm having rod shaped particles, a particle size distribution of about 20 Mu to about 60 Mu and a water regain of 2.3 to 2.7 gm./gm. exchanger; j. collecting the eluates resulting from eluting with ammonium acetate at pH 4 to 0.1 M sodium chloride.

Claim 8

A process for producing human carcinoembryonic antigen component B which comprises a. homogenizing adenocarcinoma tissue; b. separating the solid particles from the homogenate; c. treating the supernatant with a glycoprotein solvent in which the carcinoembryonic antigen component B is soluble; d. separating the precipitate and clarifying the resulting solution; e. dialyzing the clarified solution against a polyethylene glycol with an average molecular weight of about 15,000 to 20, 000 and a softening point at 60*C.; f. dissolving the retentate in an aqueous buffer of pH from about 5 to 9; g. subjecting the resulting material to sequential chromatography on two different gel columns, the first of which is an agarose gel having about 6 percent by weight agarose and a particle size of from 40 to 210 microns and the second of which is a hydrophilic water-insoluble cross-linked dextran polymer gel; h. collecting and concentrating the eluates which have a spectrophotometric absorption peak wave length of 280 m Mu and a molecular weight of about 200,000 to 500,000; i. subjecting the resulting eluates to chromatography on a mixed bed cellulose ion exchange column comprising microgranular diethylaminoethyl cellulose in the free base form having rod shaped particles, a capacity of 1.0 + or - 0.1 meq./gm., a particle size distribution of about 20 Mu to about 60 Mu and a water regain of 2.3 to 2.8 gm./gm. dry exchanger and a microgranular carboxymethycellulose with a capacity of 1.0 + or - 0.1 meq./gm having rod shaped particles, a particle size distribution of about 20 Mu to about 60 Mu and a water regain of 2.3 to 2.7 gm./gm. exchanger; j. collecting the eluates resulting from eluting with ammonium acetate at pH 4 to 0.1 M sodium chloride.

Claim 9

The process of claim 6 wherein in step (g) the material is first dissolved in an aqueous buffer of about pH 7 then run through the agarose gel column which separates the CEA material, component A or component B containing fraction from higher and lower molecular weight material and colloidal particles, the eluates having a molecular weight ranging from about 200,000 - 500,000 and a reading with a peak at 280 m Mu on a UV spectrophotometer are collected, dialyzed against polyethylene glycol of molecular weight about 20,000, the retentatee dissolved in an aqueous buffer of pH about 7 and run through a cross-linked dextran gel column which has a higher resolving power than the first column and separates CEA material, component A or component B containing fraction from higher and lower molecular weight material.

Claim 10

The process of claim 6 wherein in step (i) the eluate is dialyzed against polyethylene glycol of molecular weight of about 20,000, the retentate dissolved in an aqueous buffer of pH about 4, then run through said mixed bed ion exchange column.

Claim 11

The process of claim 6 wherein in step (i) the eluate is dialyzed against polyethylene glycol of molecular weight of about 20,000, the retentate is dissolved in an aqueous ammonium acetate buffer with 0.1 M acetate of pH about 4, then run through said mixed bed ion exchange column.