lmmunochemistry, ~/977,
Vol. 14, pp. 329-336.
P e r g a m o n Press.
P r i n t e d in G r e a t B r i t a i n
I M M U N O A D S O R B E N T P U R I F I C A T I O N OF THE CARCINOEMBRYONIC ANTIGEN L E O N I E K. A S H M A N * and NEVILLE J. DE Y O U N G Department of Surgery, University of Adelaide. Adelaide 5000, South Australia (First received 6 December 1976: in revised form 17 January 1977)
Abstraet--Carcinoembryonic antigen (CEA), a carbohydrate-rich glycoprotein, has been purified from human tumour tissue by three methods: (i) the procedure of Krupey et al. (1972) involving extraction with 1 M perchloric acid; (ii) by immuno-affinity chromatography using anti-CEA antibodies coupled to Sepharose 4B; and (iii) by affinity chromatography on Concanavalin A Sepharose. The immunoaffinity chromatography method was rapid and gave good yields of highly purified product, and the immuno-adsorbent could be used for many purification cycles. CEA is heterogeneous with respect to its binding to Concanavalin A-Sepharose. The major component was purified 27-fold using this affinity adsorbent, but the product still contained considerable amounts of impurities. CEA's purified from the same source by immuno-affinity chromatography and by the perchloric acid extraction method had similar mol. wts and electrophoretic mobility. However the immunoadsorbent-purified material had a higher ratio of carbohydrate to protein, 64.3°,o compared to 50.3?/o, implying that acid extraction resulted in partial degradation of the carbohydrate moiety. The two preparations were immunochemically similar but not identical.
INTRODUCTION Carcinoembryonic antigen (CEA)t originally identified in human tumour tissue extracts by Gold and Freedman (1965), is a high tool. wt glycoprotein containing approx 50% carbohydrate by weight (Terry et al., 1974). The usual purification procedure for CEA (Krupey e t al., 1972) involves extraction of a tumour homogenate with 1 M perchloric acid, a process which we have now shown results in a loss of carbohydrate from the molecule. Studies of the presently unknown biological function of CEA. require a more gentle purification method. During our initial attempts to purify CEA without the use of perchloric acid, we noted considerable charge heterogeneity as evidenced by elution from D E A E Sephadex of multiple peaks reactive with specific anti-CEA serum. Similar charge heterogeneity of CEA has been reported by other workers (Coligan et al., 1973; Rule & Goleski-Reilly, 1974; Eveleigh, 1974; Banjo et al., 1974). Some size heterogeneity has also been found (Coligan et al., 1973; Newman et al., 1974). Such molecular heterogeneity, which commonly occurs in carbohydrate-rich glycoproteins due to the nature of their synthesig (Gottschalk. 1969), renders ineffective many of the usual biochemical procedures for purification and assessment of purity of proteins. Methods based on specific affinity can be effectively used in the purification of macromolecules displaying molecular heterogeneity. We have corn* Present address: Department of Microbiology and Immunology, University of Adelaide, Adelaide 5000, South Australia. t Abbreviations used: CEA, carcinoembryonic antigen; PAS, periodic acid-Schiff reagent. :~ International reference preparation of carcinoembryonic antigen (Lot 73/601) was obtained from the British Medical Research Council, National Institute for Biological Standards and Control, Holly Hill, London, U.K. 329 I\l~l h4 •
I~
pared two such methods, affinity chromatography on Concanavalin A Sepharose and immuno-affinity chromatography, with the perchloric acid extraction method for purification of CEA.
MATERIALS AND METHODS Preparation of standard CEA and antiserum
CEA for use as our laboratory standard and for raising antiserum, was prepared from hepatic metastases of colonic carcinoma (obtained at autopsy) by the method of Krupey et al. (1972), as modified by Coligan et al. (1972). The procedure involved extraction with 1 M perchloric acid and gel filtration on Sepharose 4B, then Sephadex G200. The product gave a single arc on immunoelectrophoresis with antiserum to a crude perchloric acid extract of the tumour, and was immunochemically indistinguishable from the first British CEA standard~ (Laurence et al., 1975) based on the slopes of lo0t-transformed radioimmunoassay standard curves. Antisera were prepared in rabbits and goats by subcutaneous injection of purified CEA (2.0 mg for goats; 500/~g for rabbits; emulsified with Freund's complete adjuvant) at monthly intervals. Animals were bled 10-14 days after each injection. Immunoadsorbent preparation
The IgG fraction from high titre immune sera, partially purified by (NH4)2SO 4 fractionation (Stelos, 1967), was coupled to cyanogen bromide activated Sepharose 4B in 0.2 M citrate (Na ~, pH 6.5) as described by Cuatrecasas and Afinsen (1971) at a protein concentration of 10 mg/ml gel. The antigen binding capacity of the immunoadsorbent was determined by incubating 0.1 ml aliquots with serial 1/2 dilutions of trace 12SI-labelled CEA in a final volume of 1.0 ml on a rotator for 3 hr at 2Y'C and measuring the radioactivity in supernatant and the washed gel. The amount of antigen which could be 50~o bound by the gel was obtained by interpolation. The antigen capacity per 0.1 ml of gel was taken as half of that amount.
330
LEONIE K. ASHMAN and NEVILLE ,I. DE YOUNG
lmmtlmmdsorbent l~ltv(li95'~.; precipitable by monospecific anti-CEA serum. No Coomassie blue staining bands were detected in the perchloric acidpurified o r immunoadsorbent-purified CEA preparations, but the product of the single-step purification on Concanavalin A Scpharose gave a number of Coomassie blue staining bands which presumably represent contaminating glycoproteins of low carbohydrate content. Polyacrylamide gel electrophoresis was, however, a
poor indicator of purity of CEA. When the perchloric acid purification procedure was monitored using the Ornstein-Davis system (Fig. 2) no difference in banding pattern was discernable between the product of the first gel filtration step and the final product. although there was a 5-fold increase in immunological activity' per nnit weight (Table 21, hnpurities were more readily detected by iinmunoelectrophoresis using antiserum prepared by immunising rabbits with a crude glycoprotein fraction from colonic tumour tissue. By this method, an impurity could be detected in perchloric acid extracted CEA purified to the Sephadex (}200 step (Fig. 2j. Thns, provided antiserum of appropriately broad specificity is used. immunoeleetrophoresis may be a more sensitive method for assessing the purity of micro-heterogenious glycoproteins such as CEA, Precipitin arcs obtained with CEA were. however, non-idea/. This presumably also rellects the micro-heterogeneity of the antigen since it could not bc overcome by varying the ratio of antiserum to antigen. On immunoelectrophoresis immunoadsorbent-purified CEA, like conventionally purified CEA, gave a single precipitin arc with broad specificity antiserum (Fig. 4). Multiple arcs were obtained using the product from single-step purification on Concanavalin A Sepharose indicating considerable impurity. Therefore. the properties of this preparation were not further studied.
Properties o[ immunoadsorhent-pur!fied and perchloric acid-lnW(lied CEA As already indicated, CEA purified by the immunoadsorbent method and by the perchloric acid extraction method used by other workers had similar
lmmunoadsorbent Purification of CEA
333
ta"
{b) 1
2
Fig. 3. Polyacrylamide gel electrophoretic comparison of CEA after purification by, the three different methods. (a) CEA prepared by the conventional method (lJ, CEA prepared by immunoadsorbent chromatography (2), and CEA prepared by a single chromatography step on ConA Sepharose (3) were run in the Ornstein-Davis system and stained with PAS. (b) As above, and stained with Coomassie blue. gross molecular properties as indicated by their behaviour on immunoelectrophoresis and polyacrylamide gel electrophoresis. In addition, both preparations eluted from Sephadex G200 as a single peak with v/v0 of 1.2 indicating similar tool. wts. The amino acid and sugar compositions of the two preparations are shown in Tables 3 and 4. The amino acid compositions of the two preparations are very similar to each other and to that of the 1st British Standard CEA (Laurence et al., 1975). However, the immunoadsorbent purified material contained a higher proportion of carbohydrate, 64.3~ by weight, as compared with 50.3~ for perchloric acid-purified CEA. The perchloric acid-purified material contained smaller amounts of all sugars with the decrease in mannose being the most striking (Table 4). Immunochemical properties of the two preparations were compared by radioimmunoassay using antiserum to perchloric acid-purified CEA, Curves for displacement of 125I-CEA from a limiting amount of antibody as a function of the log of the amount of
unlabelled antigen added are shown in Fig. 5. The slight but signifcant (p < 0.01) difference in the slopes of the logit transformed inhibition curves (inset) indicates thatthe two preparations were immunochemically similar but not identical. DISCUSSION
The immunoadsorbent approach provides a rapid, single-step method for obtaining CEA of high purity and in good yield. It should facilitate purification and characterization of CEA from poor sources (such as normal colonic mucosa), which is extremely difficult by the more usual method involving perchloric acid extraction. In addition, it avoids the extreme oxidising and acid conditions of perchloric acid extraction, which appears to cause some loss of carbohydrate from the molecule, and should therefore yield a product more suitable for studies of the biological function of CEA. Immunoadsorbent-purified CEA differed only slightly from perchloric acid-purified CEA in immunochemical properties despite its considerably
334
LEONIt: K. ASHMAN and NEVILLf2 ,1. DE YOI!N(I !
!
Ib
2
"
gGt
3 t
'*"
4 I
I
t Fig. 4. lmmunoelcctrophoretic comparison of CEA after purification by the threc different methods. (t) Crude tmnotn" extracL 12) CEA prepared by the conventional method. (3) ('EA prepared by a single chromatography step on ConA Sepharose. and (4) ('EA prepared by immunoadsorbcnt chromatography, were tested with an antiserum raised against a crude glycoprotein fi-action from hepatic metastases from a colonic carcinoma. Conditions of immunoclecirophorcsis v~crc as described in the legend to Fig. 2.
higher c a r b o h y d r a t e content. This is in accord with the finding of H a m m a r s t r ~ m e t a / . (1975) that the antigenic d e t e r m i n a n t s of C F A reside primarily in lhe peptide moiety. Affinity c h r o m a t o g r a p h y on ( ' n n c a n a w l l i n A S e p h a r o s e as the initial puritication step gave a
27-fold purification o f C E A and provides a simple a n d gentle alternative to perchloric acid extraction for the removal of the bulk of the protein. However. the p r o d u c t eluted from C o n c a n a v a l i n A. S e p h a r o s e contained only about 16",, C E A by weight, and further fractionation is required to give a pure p r o d -
Table 3 A m i n o acid conlposition el ('t{,,\ prcparalions hllmunoadsorbenipuriticd
tt(l():
ptllli[ied 'F\
{I/IoIC
(I ~ ~,l hq;ll ;111]111~1;Ic'icl'q
("1: ,%/'
Lysino Histidine Arginine Aspartatc Th reon ira:
2.(~ 2.n 3.2 145) 9.0
~. I
9.7
Sermc Gltitanlate Proiine
11.4 [l).2
1l) 5 I[). 5
1[/.4 [O.g
5.3 ~.4 h.4 7.2 6.7 7.3 3.9 t~. I 4. I 2.S N.D. N.I).
~.7
G lycinc Alaninc Valine Isoleucine Leucine T) rosinc Phenylalanhac
Half cystemc Methioninc
4.4 1.
Vol. 14, pp. 329-336.
P e r g a m o n Press.
P r i n t e d in G r e a t B r i t a i n
I M M U N O A D S O R B E N T P U R I F I C A T I O N OF THE CARCINOEMBRYONIC ANTIGEN L E O N I E K. A S H M A N * and NEVILLE J. DE Y O U N G Department of Surgery, University of Adelaide. Adelaide 5000, South Australia (First received 6 December 1976: in revised form 17 January 1977)
Abstraet--Carcinoembryonic antigen (CEA), a carbohydrate-rich glycoprotein, has been purified from human tumour tissue by three methods: (i) the procedure of Krupey et al. (1972) involving extraction with 1 M perchloric acid; (ii) by immuno-affinity chromatography using anti-CEA antibodies coupled to Sepharose 4B; and (iii) by affinity chromatography on Concanavalin A Sepharose. The immunoaffinity chromatography method was rapid and gave good yields of highly purified product, and the immuno-adsorbent could be used for many purification cycles. CEA is heterogeneous with respect to its binding to Concanavalin A-Sepharose. The major component was purified 27-fold using this affinity adsorbent, but the product still contained considerable amounts of impurities. CEA's purified from the same source by immuno-affinity chromatography and by the perchloric acid extraction method had similar mol. wts and electrophoretic mobility. However the immunoadsorbent-purified material had a higher ratio of carbohydrate to protein, 64.3°,o compared to 50.3?/o, implying that acid extraction resulted in partial degradation of the carbohydrate moiety. The two preparations were immunochemically similar but not identical.
INTRODUCTION Carcinoembryonic antigen (CEA)t originally identified in human tumour tissue extracts by Gold and Freedman (1965), is a high tool. wt glycoprotein containing approx 50% carbohydrate by weight (Terry et al., 1974). The usual purification procedure for CEA (Krupey e t al., 1972) involves extraction of a tumour homogenate with 1 M perchloric acid, a process which we have now shown results in a loss of carbohydrate from the molecule. Studies of the presently unknown biological function of CEA. require a more gentle purification method. During our initial attempts to purify CEA without the use of perchloric acid, we noted considerable charge heterogeneity as evidenced by elution from D E A E Sephadex of multiple peaks reactive with specific anti-CEA serum. Similar charge heterogeneity of CEA has been reported by other workers (Coligan et al., 1973; Rule & Goleski-Reilly, 1974; Eveleigh, 1974; Banjo et al., 1974). Some size heterogeneity has also been found (Coligan et al., 1973; Newman et al., 1974). Such molecular heterogeneity, which commonly occurs in carbohydrate-rich glycoproteins due to the nature of their synthesig (Gottschalk. 1969), renders ineffective many of the usual biochemical procedures for purification and assessment of purity of proteins. Methods based on specific affinity can be effectively used in the purification of macromolecules displaying molecular heterogeneity. We have corn* Present address: Department of Microbiology and Immunology, University of Adelaide, Adelaide 5000, South Australia. t Abbreviations used: CEA, carcinoembryonic antigen; PAS, periodic acid-Schiff reagent. :~ International reference preparation of carcinoembryonic antigen (Lot 73/601) was obtained from the British Medical Research Council, National Institute for Biological Standards and Control, Holly Hill, London, U.K. 329 I\l~l h4 •
I~
pared two such methods, affinity chromatography on Concanavalin A Sepharose and immuno-affinity chromatography, with the perchloric acid extraction method for purification of CEA.
MATERIALS AND METHODS Preparation of standard CEA and antiserum
CEA for use as our laboratory standard and for raising antiserum, was prepared from hepatic metastases of colonic carcinoma (obtained at autopsy) by the method of Krupey et al. (1972), as modified by Coligan et al. (1972). The procedure involved extraction with 1 M perchloric acid and gel filtration on Sepharose 4B, then Sephadex G200. The product gave a single arc on immunoelectrophoresis with antiserum to a crude perchloric acid extract of the tumour, and was immunochemically indistinguishable from the first British CEA standard~ (Laurence et al., 1975) based on the slopes of lo0t-transformed radioimmunoassay standard curves. Antisera were prepared in rabbits and goats by subcutaneous injection of purified CEA (2.0 mg for goats; 500/~g for rabbits; emulsified with Freund's complete adjuvant) at monthly intervals. Animals were bled 10-14 days after each injection. Immunoadsorbent preparation
The IgG fraction from high titre immune sera, partially purified by (NH4)2SO 4 fractionation (Stelos, 1967), was coupled to cyanogen bromide activated Sepharose 4B in 0.2 M citrate (Na ~, pH 6.5) as described by Cuatrecasas and Afinsen (1971) at a protein concentration of 10 mg/ml gel. The antigen binding capacity of the immunoadsorbent was determined by incubating 0.1 ml aliquots with serial 1/2 dilutions of trace 12SI-labelled CEA in a final volume of 1.0 ml on a rotator for 3 hr at 2Y'C and measuring the radioactivity in supernatant and the washed gel. The amount of antigen which could be 50~o bound by the gel was obtained by interpolation. The antigen capacity per 0.1 ml of gel was taken as half of that amount.
330
LEONIE K. ASHMAN and NEVILLE ,I. DE YOUNG
lmmtlmmdsorbent l~ltv(li95'~.; precipitable by monospecific anti-CEA serum. No Coomassie blue staining bands were detected in the perchloric acidpurified o r immunoadsorbent-purified CEA preparations, but the product of the single-step purification on Concanavalin A Scpharose gave a number of Coomassie blue staining bands which presumably represent contaminating glycoproteins of low carbohydrate content. Polyacrylamide gel electrophoresis was, however, a
poor indicator of purity of CEA. When the perchloric acid purification procedure was monitored using the Ornstein-Davis system (Fig. 2) no difference in banding pattern was discernable between the product of the first gel filtration step and the final product. although there was a 5-fold increase in immunological activity' per nnit weight (Table 21, hnpurities were more readily detected by iinmunoelectrophoresis using antiserum prepared by immunising rabbits with a crude glycoprotein fraction from colonic tumour tissue. By this method, an impurity could be detected in perchloric acid extracted CEA purified to the Sephadex (}200 step (Fig. 2j. Thns, provided antiserum of appropriately broad specificity is used. immunoeleetrophoresis may be a more sensitive method for assessing the purity of micro-heterogenious glycoproteins such as CEA, Precipitin arcs obtained with CEA were. however, non-idea/. This presumably also rellects the micro-heterogeneity of the antigen since it could not bc overcome by varying the ratio of antiserum to antigen. On immunoelectrophoresis immunoadsorbent-purified CEA, like conventionally purified CEA, gave a single precipitin arc with broad specificity antiserum (Fig. 4). Multiple arcs were obtained using the product from single-step purification on Concanavalin A Sepharose indicating considerable impurity. Therefore. the properties of this preparation were not further studied.
Properties o[ immunoadsorhent-pur!fied and perchloric acid-lnW(lied CEA As already indicated, CEA purified by the immunoadsorbent method and by the perchloric acid extraction method used by other workers had similar
lmmunoadsorbent Purification of CEA
333
ta"
{b) 1
2
Fig. 3. Polyacrylamide gel electrophoretic comparison of CEA after purification by, the three different methods. (a) CEA prepared by the conventional method (lJ, CEA prepared by immunoadsorbent chromatography (2), and CEA prepared by a single chromatography step on ConA Sepharose (3) were run in the Ornstein-Davis system and stained with PAS. (b) As above, and stained with Coomassie blue. gross molecular properties as indicated by their behaviour on immunoelectrophoresis and polyacrylamide gel electrophoresis. In addition, both preparations eluted from Sephadex G200 as a single peak with v/v0 of 1.2 indicating similar tool. wts. The amino acid and sugar compositions of the two preparations are shown in Tables 3 and 4. The amino acid compositions of the two preparations are very similar to each other and to that of the 1st British Standard CEA (Laurence et al., 1975). However, the immunoadsorbent purified material contained a higher proportion of carbohydrate, 64.3~ by weight, as compared with 50.3~ for perchloric acid-purified CEA. The perchloric acid-purified material contained smaller amounts of all sugars with the decrease in mannose being the most striking (Table 4). Immunochemical properties of the two preparations were compared by radioimmunoassay using antiserum to perchloric acid-purified CEA, Curves for displacement of 125I-CEA from a limiting amount of antibody as a function of the log of the amount of
unlabelled antigen added are shown in Fig. 5. The slight but signifcant (p < 0.01) difference in the slopes of the logit transformed inhibition curves (inset) indicates thatthe two preparations were immunochemically similar but not identical. DISCUSSION
The immunoadsorbent approach provides a rapid, single-step method for obtaining CEA of high purity and in good yield. It should facilitate purification and characterization of CEA from poor sources (such as normal colonic mucosa), which is extremely difficult by the more usual method involving perchloric acid extraction. In addition, it avoids the extreme oxidising and acid conditions of perchloric acid extraction, which appears to cause some loss of carbohydrate from the molecule, and should therefore yield a product more suitable for studies of the biological function of CEA. Immunoadsorbent-purified CEA differed only slightly from perchloric acid-purified CEA in immunochemical properties despite its considerably
334
LEONIt: K. ASHMAN and NEVILLf2 ,1. DE YOI!N(I !
!
Ib
2
"
gGt
3 t
'*"
4 I
I
t Fig. 4. lmmunoelcctrophoretic comparison of CEA after purification by the threc different methods. (t) Crude tmnotn" extracL 12) CEA prepared by the conventional method. (3) ('EA prepared by a single chromatography step on ConA Sepharose. and (4) ('EA prepared by immunoadsorbcnt chromatography, were tested with an antiserum raised against a crude glycoprotein fi-action from hepatic metastases from a colonic carcinoma. Conditions of immunoclecirophorcsis v~crc as described in the legend to Fig. 2.
higher c a r b o h y d r a t e content. This is in accord with the finding of H a m m a r s t r ~ m e t a / . (1975) that the antigenic d e t e r m i n a n t s of C F A reside primarily in lhe peptide moiety. Affinity c h r o m a t o g r a p h y on ( ' n n c a n a w l l i n A S e p h a r o s e as the initial puritication step gave a
27-fold purification o f C E A and provides a simple a n d gentle alternative to perchloric acid extraction for the removal of the bulk of the protein. However. the p r o d u c t eluted from C o n c a n a v a l i n A. S e p h a r o s e contained only about 16",, C E A by weight, and further fractionation is required to give a pure p r o d -
Table 3 A m i n o acid conlposition el ('t{,,\ prcparalions hllmunoadsorbenipuriticd
tt(l():
ptllli[ied 'F\
{I/IoIC
(I ~ ~,l hq;ll ;111]111~1;Ic'icl'q
("1: ,%/'
Lysino Histidine Arginine Aspartatc Th reon ira:
2.(~ 2.n 3.2 145) 9.0
~. I
9.7
Sermc Gltitanlate Proiine
11.4 [l).2
1l) 5 I[). 5
1[/.4 [O.g
5.3 ~.4 h.4 7.2 6.7 7.3 3.9 t~. I 4. I 2.S N.D. N.I).
~.7
G lycinc Alaninc Valine Isoleucine Leucine T) rosinc Phenylalanhac
Half cystemc Methioninc
4.4 1.
