The detection of carcinoembryonic antigen in whole serum from patients with malignant and nonmalignant disease

CLINICAL

IhlMUNOLOGY

AND

IMhlUNOPATHOLOGY

The Detection in Whole Malignant J. M. MACSWEEN~, The W(lltrr

1,

330-345

(1973)

of Carcinoembryonic Antigen Serum from Patients with and Nonmalignant Disease1 N. L. WAHNER

trr~d Elix Hall Mclhrcr~w,

AND I. R. MACYAY~

Irutitute qf Medical Arrstrulici

Heseurch,

A radioimmunoassay for carcinoembryonic antigen (CEA) in human serum was developed using small samples of whole serum. Assays for CEA in 25 ~1 of whole serum, using precipitation of immune complexes by ammonium sulfate, failed to detect CEA in many sera due to binding of globulins to CEA. Precipitation by rabbit antiserum to goat globulin proved satisfactory as there were few false negative results in assaying sera from patients with cancer, but there were false positive assays with 10% of sera from blood donors and some 30% of sera from patients with nonmalignant diseases. Some sera, particularly from patients with pancreatitis or alcoholic cirrhosis, were still positive after extraction with perchloric acid. Sera from several groups of cancer patients were positive in high frequency, in particular cancer of the gastrointestinal tract, lung, and breast. Binding of “‘1 CEA to serum globulins was demonstrated with many sera and there was no correlation with the clinical diagnosis. The use of several different binding assays failed to show clear evidence of specific antibody type binding, thus Euggesting that nonspecific binding of ““I CEA to some immunoglobulins can occur.

INTRODUCTION Carcinoembryonic antigen (CEA) is a glycoprotein which has been extracted from carcinomas of the colon and purified by column chromatography and electrophoretic techniques (l-3). It was initially shown to be present in the serum of patients with colon cancer by the use of a radioimmunoassay which detected the antigen after its extraction from the serum with perchloric acid (4). Using this or related methods, several other groups have reported that CEA can also be found in the serum of patients with some other cancers, and also in the serum of some patients with nonmalignant disease (5-12). As the extraction of serum with perchloric acid involves tedious and time consuming methods (extraction, dialysis, and lyophilisation) we have attempted to develop a microassay which can directly detect CEA in small samples of whole serum. In this paper we describe results with this method using sera from patients with cancer or nonmalignant diseases, and compare ’ The work was supported by Research Grants from the Bushells Trust and the National Health and Medical Research Council, Canberra. This is publication No. 1765 of the Walter and Eliza Hall Institute. 2 Fellow of the Medical Research Council, Canada. Present address: Dalhousie University, Halifax, Nova Scotia, Canada. :j Address for reprints: Dr. I. R. Mackay, The Walter and Eliza Hall Institute, R. M. H., Post Office, Parkville, 3050, Australia.

Copyright All rights

@ 1973 by Academic Press, Inc. of reproduction in any form reserved.

330

CARCINOEMBRYONIC

ANTIGEN

IN WHOLE

SERUM

331

the use of ammonium sulfate with antibody to goat gamma globulin in precipitating the CEA-anti CEA complex. With the use of ammonium sulfate, an indication was found of binding of CEA to a component in whole serum. Results with several radioimmunoassays are also presented which attempt to determine whether some patients may have antibody to CEA in their serum. MATERIALS

AND

METHODS

Radioimmunoassay for CEA in serum. A detailed description of the microradioimmunoassay employed will be presented elsewhere.4 Briefly the technique is as follows: Purified CEA and goat antiserum to CEA were generously provided by Dr. P. Gold, Montreal; 2-20 pg samples of CEA were labeled with lzsI using chloramine T as oxidant (13); the specific activity of various preparations was 22-50 &i/pg. Labeled CEA and goat antiserum to CEA were diluted in 0.01 M Tris buffer pH7.5 containing 3% bovine serum albumin and were stored at -20°C. Several weeks after iodination, the labeled antigen became less precipitable, and the nonprecipitable component was removed by gel filtration on Sephadex G-200. Full activity was found in the first peak just back of the void volume. Each serum assay was performed in duplicate in 6 x 50 mm tubes by mixing 25 ~1 of patients serum with 25 ~1 of either a 1 X 10e5or 5 X lo+ dilution of goat antiserum to CEA; the tubes were held for 2 hr at 37°C and then overnight at 4°C; 50 ~1 of lz51CEA (concentration 1 rig/ml) was then added and the tubes again held for 2 hr at 37°C and overnight at 4°C. Control tubes included (1) 1251CEA with pooled normal human serum, (2) 1251CEA with goat antiserum to CEA and pooled normal human serum, and (3) 1251CEA with goat antiserum to CEA, normal human serum, and doubling dilutions of purified unlabeled CEA at concentrations of 3-100 rig/ml, to obtain a standard inhibition curve. Precipitation of antigen-antibody complexes was carried out either with ammonium sulfate or antiserum to goat gamma globulin. In the former case, an equal volume (100 ~1) of saturated ammonium sulfate was added to each tube, immediately mixed, held for 1 hr at 4°C centrifuged at 16,000g for 10 min, and a 100 ~1 sample of the supernate from each tube removed for counting of radioactivity. In the alternative procedure, 100 ~1 of antiserum to goat, gamma globulin derived from sheep (Meloy laboratories) or rabbit (Hoechst, Australia, Ltd.), in a dilution of 1 X 10-l or 2 x lop2 was added to each tube, held at 37°C for 2 hr and overnight at 4°C centrifuged at 16,OOOg and the supemates were counted as described above. Perchloric acid extraction of serum. Sera were extracted using 0.1 ml of 4 M perchloric acid added to 0.4 ml of serum whilst the serum was being agitated on a vortex mixer. The tubes were centrifuged at 4000g for 10 min and the supemate dialysed against multiple changes of 0.01 M phosphate buffered saline pH7.3 for 2 days. Samples of 25 ~1 of the dialysed extracts were assayed * Warner,

N. L. and MacSween,

J. M. in preparation.

332

MACSWEEN,

WARNER

AND

MACKAY

for CEA with precipitation of immune complexes by ammonium sulfate. Precipitation was facilitated by the addition to the goat antiserum to CEA, of normal goat serum in a dilution of 2 X IOp2. Control tubes contained perchloric acid extracts of pooled normal human serum. A standard inhibition curve was obtained by including with each assay perchloric acid extracts of pooled normal human serum to which had been added before extraction doubling dilutions of unlabeled CEA. Effect of heat treatment. Serum samples of 200 ~1 were diluted with an equal volume of normal saline and heated in a boiling water bath for 30-45 set, and then centrifuged in 6 X 50 mm tubes at 36,000~ for 20 min. Assays for CEA were performed on 25 ~1 samples of supernate with precipitation of complexes by rabbit antiserum to goat globulin. Precipitation was facilitated by the addition of normal goat serum in a dilution of 2 x 10e2. Serum absorptions. Twenty-five microliters of human sera were mixed with 5 ~1 of a 1.0 X IO-’ dilution of normal rabbit serum diluted in Tris-BSA buffer, held at 37°C for 2 hr and overnight at 4°C. The sera were then assayed directly for CEA as described above using rabbit antiserum to goat globulin to precipitate immune complexes. Absorption with normal goat serum was carried out similarly using 5 ~1 of a 6.2 x 10m3 dilution of normal goat serum. Binding of human serum globulins to CEA. Duplicate samples of 20 ~1 of human sera or of human y-globulin, 20 mg/ml (Commonwealth Serum Laboratories) were diluted with 30 ~1 of Tris-BSA buffer, and held at 37°C for 2 hr and overnight at 4°C. Control tubes contained 20 ~1 of pooled normal human serum: 50 ~1 of lz51 CEA (approx 1 rig/ml) was added to each tube, held again at 37°C for 2 hr and overnight at 4°C. Saturated ammonium sulfate (100 ,ul) was added to each tube, held at 4°C for 1 hr, centrifuged at 16,OOOg for 10 min and 100 ~1 removed from each tube for counting of radioactivity. Inhibition of binding by unlabeled CEA was assessed by adding 5 ~1 of unlabeled CEA (1 pg/ml) to 25 ~1 of Tris-BSA buffer before the addition of serum. In some tests, ammonium sulfate was not used and instead complexes were precipitated by the addition of 100 ~1 of neat rabbit antiserum to human immunoglobulin to each tube which were held at 37°C for 2 hr and at 4°C overnight before centrifugation. Radioim,munoelectrophoresis. Glass slides (3 x 1 in.) were coated with 1% noble agar (Difco Laboratories, Detroit) in Verona1 buffer pH8.2. Wells and troughs were cut in the agar with a LKB gel punch and approximately 5 ~1 samples of human sera were deposited in the wells. Electrophoresis was carried out at 275 V for 85 min, after which 30 ~1 of rabbit anti-human globulin antiserum containing 30 ng of lz51 CEA was added to each trough. The slides were held at room temperature in a moist chamber for 24 hr and then washed for 48 hr with multiple changes of saline and distilled water. The slides were dried in air and the precipitation arcs stained with amido black. Autoradiographs were obtained on Kodak BB54 X-ray film placed in immediate apposition to the slides at 4°C and developed after 2 wk. Source of seru. Serum samples were obtained from 51 blood donors and 22 women in the first 6 mo of pregnancy. Selected sera from patients in the Royal Melbourne Hospital included a variety of cancers and nonmalignant diseases,

CABCINOEMBRYONIC

ANTIGEN

IN

WHOLE

333

SERUM

particularly involving the liver, pancreas, and gastrointestinal tract. Patients with carcinoma of the colon were divided into those with either local or metastatic dissemination of the tumor. (“Local” refers to Dukes’ class 1, and “disseminated” includes Dukes’ class 2 and 3, and distant metastases. Other cancers were not staged.) RESULTS Detection

of CEA in Whole

Serum

(i) Precipitation of immune complexes by ammonium sulfate. In the initial series of studies competitive inhibition assays were performed using 25 ~1 of whole serum, 50 ~1 of labeled CEA, and 25 ~1 of goat anti CEA serum at a suitable dilution followed a day later by an equal volume of saturated ammonium sulfate. In the absence of any added serum or inhibitor, approximately 30% precipitation of labeled antigen was observed. However if normal human serum or gamma globulin was also added, increased precipitation of lz51 labeled CEA was frequently observed, and some precipitation was observed even in the absence of added goat antiserum to CEA suggesting binding of serum components to lz51 CEA (Table 1). This result was in contrast to that observed when sera from many colon cancer patients were added, when decreased precipitation of labeled CEA by goat antiserum to CEA resulted (i.e., inhibition of precipitation). The former result raised the likelihood that in sera containing a low but definite amount of CEA, the binding capacity for labeled CEA of whole serum may mask the inhibitory activity of the CEA in the immunoassay. To test this, the inhibitory capacity of normal human serum containing added CEA was determined, using eight normal sera, of which four gave binding of CEA in the absence of anti-CEA antibody, and four did not. The results (Table 2) were as follows. In the absence of any added inhibitor or serum, the amount of goat anti CEA added resulted in 42% precipitation of

PRECIPITATION

OF rzJI CEA

TABLE BY NORMAL

1 GAMMA Percent

Proteins Nil HGGb NHS’

added

No goat anti-CEA 0 19 12

GLORULIN precipitation

AND of Y

SERUM CEA” Goat

anti-CEA 36 43 43

a Precipitation of labeled CEA by added protein in the presence (as a final dilution of 5 X 10e6) or absence of added goat antibody to CEA. Complexes precipitated by half saturated ammonium sulfate. b Human gamma globulin (Commonwealth Serum Laboratories, Melb) at a concentration of 20 mglml. ’ Average value for 23 blood donor sera at a final dilution of 1:4.

334

MACSWEEN,

WARNER

TABLE INHIHTORY

AND

MACKAY

2

OF BLOOD DONOR SEW CONTAINING ,~IKIED CEA

ACTIVITY

Serum no.

c/c Precipitation of lz31 CEA by serum alone

Nil 1 2 3 4 5 6 7 8

% Precipitation of 12sI CEA by goat-anti CEA in the presence or absence of serum containing added CEA”,” 0

50

100

< 10 Cl0 Cl0 < 10
42 41 43 40 41

(’ 10 -c 10 II < 10 -c 10

<: 10 < 10 i 10 .‘I0 c 10

33 33 26 34

58 60 58 57

0 Serum added at final dilution of 1:4. Complexes precipitated sulfate. b Goat anti-CEA antibody at final dilution of 5 x 10~“. Added either 0, 50, or 100 rig/ml CEA.

47 %59 SO 52 by half

saturated

human

serum

40 55 47 48 ammonium also

contains

labeled CEA: with 25 ~1 of sera l-4, this amount of precipitation was not altered, whereas sera 5-8, which alone precipitated about 30% of CEA, inincreased the level of precipitation of labeled CEA to about 60%. The prior addition of either 50 or 100 rig/ml of CEA to the first four sera led to a complete inhibition of the precipitation of 125ICEA by goat anti-CEA serum. Addition of CEA to sera 5-8 resulted in all inhibitory activity of the CEA being lost (40-60% precipitation of lz51 CEA was still observed). In all of the above tests, the lz51CEA-goat anti-CEA complex was precipitated by the addition of an equal volume of saturated ammonium sulfate. As ammonium sulfate precipitates complexes of lz51CEA and serum proteins as well 9 CEA-goat anti-CEA, our results suggest that there are some proteins in gman sera, possibly including gamma globulin (see below) which bind to CEA thereby masking its inhibitory effect. (ii) Precipitntion of immune complexes by untiserum to gout gummu g/obulin. To overcome this problem encountered with the use of whole serum and ammonium sulfate, we studied the effect of specific antisera to goat gamma globulin in precipitating the lz51 CEA-goat anti-CEA complex. Normal sera having marked binding to lz51CEA were tested for their ability to inhibit lz51 CEA-goat anti-CEA precipitation: tests were made with normal sera, and normal sera with added unlabeled CEA, immune complexes were precipitated by adding either ammonium sulfate, or rabbit or sheep antisera to goat gamma globulin. The results given in Table 3, are shown as the CEA content of the sera as determined by comparison with a standard CEA inhibition curve. There was considerable reduction in the expected value when ammonium sulfate was used for precipitation, but only slight reduction,

CARCINOEMBRYONIC

ANTIGEN TABLE

IN

WHOLE

335

SERUM

3

INHIBITORY ACTIVITY OF BLOOD DONOR SERA CONTAINING ADDED CEA: COMPLEX PRECIPITATION BY ANTIGLOBULINS Assayed serum concentration in sera containing added Human

Precipitation complex

seruma 456 247 445 245

Amm. Amm. Amm. Amm.

450 247 445 245 450 247 445 245

of CEA CEA -

of 20’

0

Sulb Sulb Sul.* Sul.*

40

<3 <3 <3 <3

4 <3 <3 <3

12 12 25 <3

RAGG’ RAGGc RAGGc RAGG’

<3 <3 <3 <3

25 13 12 14

45 35 34 34

SAGGd SAGGd SAGGd SAGGd

<3 <3 <3

30 23 18 16

45 30 25 23

4

a All sera added at final dilution of 1:4. * Half saturated ammonium sulfate. ’ Rabbit anti-goat gammaglobulin. d Sheep anti-goat gammaglobulin. e Human sera containing previously added

CEA

at concentration

of either

20 or 46 rig/ml

60-100% of the expected, when either antiglobulin serum was used for precipitation. As these results clearly indicated that an antiglobulin precipitation method was preferable to the use of ammonium sulfate for assay of CEA in whole serum, we therefore determined the effect of several minor variations in the antiglobulin method, so as to use an optimal assay for the inhibitory studies to be performed. The results in Table 3 indicated that both sheep and rabbit antisera to goat gamma globulin were suitable for precipitation. However, when a wider selection of normal sera were tested for inhibitory activity, without any added CEA, it was found with assays employing sheep antiserum that many normal sera gave inhibition of precipitation that was equivalent to a concentration of CEA of 5-15 rig/ml (Table 4). This was not observed as frequently with rabbit antiserum to goat gamma globulin so that in all further assays only rabbit antiserum was used. TABLE

4

ASSAY FOR CEA IN BLOOD DONOR SERA USING EITHER RABBIT OR SHEEP ANTI-COAT GAMMA GLOBULIN FOR PRECIPITATION Antiserum for complex precipitation Sheep Rabbit

anti-goat anti-goat

Q Results

show

gamma gamma number

Assayed

globulin globulin of sera with

serum <5

concentration 5-Q

6 13 “CEA”

concentrations

3 0 detected.

of CEA 10-20 4 0

(ng/mlp

336

MACSWEEN,

EFFECT

WARNER

AND

MACKAY

TABLE 5 OF TIME OF INCUBATION OF HUMAN SERUM WITH GOAT ANTIBODY ON INHIBITORY ACTIVITY OF SERUM

ANTI-WA

Percent inhibition of precipitation of lssI CEA and goat anti-CEA antibody by normal human serum containing added CEAb Amounts of CEA in normal human serum pool (&ml)”

No preincubation -____

0 50.0 25.0 12.5 6.2 3.1

0 21 10 5 2 0

” Concentration of CEA in normal human serum goat anti-CEA antibody. b Percentage of inhibition of precipitation of ‘9 serum and goat antibody to CEA were preincubated

2 hr 37°C 18 hr 4°C

2 hr 37°C __-..0 57 31 15 10 5

(25 ~1) added

0 78 43 25 17 10

to 25 /.LI of 1 X 10e5 dilution

CEA by serum containing or not, for time.

added

CEA,

of’ when

The order of addition of the reagents to the assay microtubes was tested (Table 5). The results indicated that maximal sensitivity of inhibition was obtained when the test serum was preincubated for about 18 hr with the goat anti-CEA antibody before the addition of lz51 CEA. (iii) Detection of CEA in whole sera derivedfionz dieerent patients. Using the conditions described in section (ii) 379 human sera were assayed for the presence of CEA (test serum preincubated for 18 hr with goat anti-CEA antibody: complexes precipitated with rabbit anti-goat y globulin) (Table 6). Ten percent (5/51) of normal blood donor sera showed inhibition of precipitation in the assays, corresponding to CEA levels of 5-14 nglml. The values being (6,8,9,12,14) rig/ml. All patient groups tested, showed a higher proportion of sera giving detectable levels of CEA. All 21 patients with disseminated cancer of the colon, and 87% of 23 with localized cancer of the colon had levels of CEA over 5 rig/ml, and 95 and 48%, respectively, had levels over 15 ngjml. Also most sex-a from patients with other cancers of the gastrointestinai tract (mainly stomach and pancreas), and from patients with lung or breast cancer, were usually positive (79-91% had levels over 5 rig/ml, and 55-600/c had levels over 15 nglml). On the other hand, only 2 of 31 sera from patients with other cancers had levels of CEA over 15 rig/ml. (These two cancers were of kidney and anus.) In general, sera from patients with nonmalignant diseases had levels of CEA, and a frequency of positive tests (over 5 rig/ml) that were intermediate between the normal blood donor population and the patients with gastrointestinal, lung, and breast cancer. Thus, 30-40% of the sera from most disease groups had levels between 5 and I4 rig/ml, but relatively few levels were over 15 nglml. The only clear exception to this was with sera from pancreatitis and

CARCINOEMBRYONIC

ANTIGEN

TABLE

LEVELS OF CEA

IN

diagnosis

Blood donors Ca colon (local) Ca colon (disseminated) Other gastrointestinal Ca Ca lung or breast Other Ca Pancreatitis or alcoholic cirrhosis” Primary biliary cirrhosis or chronic active hepatiti@ Gastritis or ulcerative colitisb Other GI tract or pulmonary diseasesb Other nonmalignant diseases ” CEA levels plexes by rabbit ’ Approximately were similar.

337

SERUM

6

IN WHOLE SERUM FROM VARIOUS PATIENT GROUPS CEA

Patient

WHOLE

No. of patients

<5

levels

in serum”

5-14

15-35

>35

Percent sera with >5 rig/ml CEA

51 23 21 34 36 31 24

46 3 0 4 5 26 5

5 9 1 10 13 3 6

0 9 10 8 7 2 12

0 2 10 12 11 0 1

10 87 100 88 86 16 79

26

19

6

1

0

27

37

26

6

5

0

30

42

25

12

5

0

40

51

32

17

2

0

37

(rig/ml) were determined on 25 ~1 of whole anti-goat gamma globulin. equal numbers of each serum type were

serum

with

tested

and

precipitation results

for each

of comgroup

alcoholic cirrhosis in whom 69 and 91%, respectively, had levels over 5 rig/ml, and 55% of each had levels over 15 rig/ml. It is of considerable interest that raised levels of CEA in sera of patients with liver disease applied to patients with alcoholic cirrhosis and mostly not to patients with primary biliary cirrhosis or active chronic hepatitis with cirrhosis. Detection

of CEA in Sera After Extraction

with Perchloric Acid

As raised CEA levels were found in the above tests with sera from many patients with nonmalignant disease, we accordingly tested many of these sera both before and after extraction with perchloric acid. One hundred and seventy-five sera were selected for extraction, which included sera from most cases of localised carcinoma of the colon, and from most cases of nonneoplastic diseases which were positive (over 5 rig/ml CEA) on-direct assay of whole serum. These extracted sera were tested in a similar microassay, but using ammonium sulfate to precipitate the complexes (the problem encountered previously with ammonium sulfate is only pertinent to the use of whole serum as test inhibitor). The assay was sufficiently sensitive to detect 3 rig/ml of CEA. As shown in Table 7, 1 of 21 normal sera, and 11 of 62 of the sera from patients with nonmalignant disease had levels of CEA above 3 rig/ml. Again the only exception in the latter category was the sera

338

MACSWEEN,

LEVELS

OF CEA

IN SELECTED

WARNER

AND

MACKAY

TABLE 7 SEFIA EXTKACTED

WITH CEA

Patient

No. of patients

diagnosis

Blood donors Ca colon (Local) Ca colon (dissemenated) Other gastrointestinal Ca Ca lung or breast Other Ca Pancreatitis or alcoholic cirrhosis Primary biliary cirrhosis or chronic active hepatitis Gastritis or ulcerative colitis Other GI tract or pulmonary disease Other nonmalignant disease u Levels from whole

of CEA in extracts serum tests.

of serum.

PEHCHLOIUC levels

Ac~u

in extracted

sera”

... 3 _._--.--..~-.

3-9

21 17 10 14 25 9 17

20 6 2 :3 7 5 4

1 7 2 3 2 2 7

0

0

3 5 I1 2 5

3 ,3 5 0 I

16

I”

.I

1

0

12 11

11 II

I 0

0 0

0 0

20

17

8

0

0

Sera

were

selected

-~-~--

on the basis

10-20 ..__--.~---

of positive

.a20 _......_-__.

reactions

from patients with pancreatitis and alcoholic cirrhosis. A high proportion of sera from patients with gastrointestional tract cancer, (65% of localized cancer of the colon) lung and breast cancer were positive. This table does not represent the overall proportion of positive sera, as it relates to sera preselected on the basis of results using whole serum. The results therefore indicate that with the exception of the sera from patients with cancer of the gastrointestinal tract, lung, and breast, and from pancreatitis, alcoholic cirrhosis, and occasionally other liver disease, most of the sera that were positive when tested directly were negative after extraction.

Elimination

of‘“Fnlse

Positive”

Results

One of the possible causes of “false positivity” (positive test for CEA on whole serum but negative test after extraction with perchloric acid) is that the patients serum may interact with either of the antibodies used in the test system, i.e., goat anti-CEA or rabbit anti-goat gamma globulin. This would reduce the concentration of the reactants, and thereby appear to inhibit precipitation. Five such false positive sera were absorbed with normal rabbit serum before assaying for CEA, but no change was observed. Prior absorption with normal goat serum was carried out on 16 “false positive” sera derived from patients with nonmalignant diseases (excluding alcoholic cirrhosis and pancreatitis). As seen in Table 8, five of these “false positive” sera became negative, three of these having had initially levels of CEA equivalent to 15 rig/ml or greater. With two of these five sera, direct binding activity to goat gamma globulin was demonstrable when the sera were mixed with izsI labeled goat gamma globulin followed by addition of rabbit antihuman gamma globulin.

CARCINOEMBRYONIC

ANTIGEN

TABLE

IN

WHOLE

339

SERUM

8

EFFECT OF ADSORPTION OF FALSE POSITIVE SERA WITH NORMAL GOAT SERUM CEA Serum

group

No. of sera

Preadsorption Adsorption with goat serumb ” Serum * Human

<5

16 16

levels detected sera preadsorbed

levels

in whole

serum”

5-15 0 5

‘: 15

12 10

4 1

with rabbit anti-goat gamma globulin precipitation of complexes. with normal goat serum at final dilution of 6 x 10m3.

As CEA is relatively stable to many physicochemical procedures, the effect of prior heating of the test inhibitory serum was studied. Assays for CEA were performed on supernates obtained after heating 22 “false positive” sera and 28 “true positive” sera from patients with cancer or pancreatitis or liver disease, Seventeen of the 22 “false positive” sera had CEA levels less than 5 rig/ml after heating (Table 9) whereas 26 of the 28 “true positive” sera remained positive after heating. These 28 sera were from patients with carcinoma of the colon, lung, and breast, and nonmalignant diseases of the liver and pancreas. Attempts to Detect Antibodies to CEA in Whole Serum by Radioimmunoassa ys The previous sections of this study have dealt with detection of CEA as antigen in whole serum. As it has been proposed (14) that human serum may also contain antibody to CEA, we attempted to demonstrate this with several types of radioimmunoassays. The results in Table 1, appeared to indicate that some sera, and even purified gamma globulin could bind to lz51CEA. Further tests with 170 sera mixed with 125TCEA, followed by ammonium sulfate precipitation failed to indicate any significant association of CEA binding activity of TABLE

9

EFFECT OF HEATING SERuhr ON CEA FALSE POSITIIX

Patient source of sera Nonmalignant

disease

Ca colon, lung, or breast Alcoholic cirrhosis or pancreatitis

CEA content in extracted sera” <3 rig/ml 5->35 ngiml

REACTIONS CEA

Pretreatment of serumh

level

nglml

No. of <5

StZl2

Nil Heated

22

Nil Heated

28

u CEA content in extracts of sera with perchloric acid. b Heating at 80°C for approx 45 sec. c CEA content in whole sera using rabbit anti-goat gamma

globulin

5-14

15-35

c35

0

14

8

0

17

3

2

0

0 2

6 8

13 8

9 10

to precipitate

complexes.

340

MACSWEEN,

PRECIPITATION

Patient

Blood donors All Cab

TABLE OF lz51 CEA

AND

Ca

and prenatalb

MACKAY

10 BY SERUM

Number of patients sera tested

diagnosis

Blood donors Prenatal Ca colon Other gastrointestinal Other Ca Nonmalignant disease

WARNER

GLOBULINS

Percent lz51 CEA --__ < 10 10-19

precipitated” 20-29

:’ 30

23 22 25 10 52 38

13 12 10 3 19 19

1 3 1 I 4 2

8 1 4 2 10 4

1 6 10 4 19 1:3

21 26

21 26

0 0

0 0

0 0

0 Number of sera showing indicated level of precipitation of rz51 CEA. after addition of equal volume of saturated ammonium sulfate. * Direct binding without addition of ammonium sulfate.

Precipitation

detected

serum with the source of the serum. The results detailed in Table 10 indicate that approximately 4O-70% of sera from many sources gave variable degrees of CEA binding, that were detected by ammonium sulfate precipitation. In order to determine whether this serum binding activity behaved in a manner analogous to the binding observed with goat anti-CEA antibody, the capacity of unlabeled CEA to inhibit the binding was ascertained. Sixty-six sera which gave over 20% binding of lz51 CEA were tested for inhibition by unlabeled CEA at a concentration equivalent to 200 rig/ml. The results in Table 11 show that binding of rz51 CEA by 19 of these sera was reduced by at least 30%. These 19 sera were then tested for their capacity to precipitate lz51 CEA when rabbit antihuman gamma globulin sera, was added rather than ammonium sulfate: only five of these sera then showed precipitation. As another sensitive procedure to detect a possible primary interaction between human immunoglobulin and 125I CEA, radioimmunoelectrophoresis was performed on 76 sera that produced over 20% binding of 125I CEA using ammonium sulfate precipitation, and on 22 sera from pregnant women. As shown in Table 12, some sera from all groups tested showed evidence of either IgM and/or IgG binding of CEA; in fact the only group of sera to show a low incidence of this activity were those from pregnant women. An example is shown in Fig. 1, in which binding of 125I CEA to immunoglobulin is seen. In evaluating the various approaches to the problem of antibody to CEA, only seldom did a serum show binding activity to CEA in all of the various assay methods. The results for the five sera that precipitated 1251CEA with the use of rabbit antihuman immunoglobulin are listed in Table 13. Although all five of these sera precipitated CEA using both ammonium sulfate and anti human globulin, and precipitation could be inhibited significantly by addition of 5 ng of CEA, only 3/5 of these sera showed binding by radioimmunoelectrophoresis, and in one case this was rather weak.

CARCINOEMBRYONIC

SPECIFICITY

ANTIGEN

OF BINDING

IN

WHOLE

TABLE 11 OF SERUM GLOBULINS

TO 9

CEA

Specificity

Patients

Total number of patients sera tested”

diagnosis

Blood donors Prenatal Ca colon Other gastrointestinal Other Ca Nonmalignant disease

Ca

Total

Inhibition unlabeled

of binding Inhibition by unlabeled CEA and precipitation by rabbit anti-HGGC

by CEAb

7 7 14 4 23 11

2 1 7 1 6 2

66

19

341

SERUM

(1 All sera gave more than 20% precipitation of iz51 CEA after addition of ammonium sulfate. b Complexes precipitated by ammonium sulfate: number of sera showing more than 30% inhibition of precipitation of 9 CEA in presence of 5 ng unlabeled CEA. c Number of sera showing both result as in (b), and giving more than 5% precipitation of Y CEA after addition of rabbit anti-human gamma globulin.

DISCUSSION The main purpose of the present study of CEA was to investigate the feasibility of assaying for CEA directly in small samples of whole serum, and second, to look for evidence of specific antibody to CEA in certain human sera. The results indicate that a microradioimmunoassay can in fact detect CEA in small samples (25 ~1) of whole serum. In the original assay system described

BINDING

TABLE 12 OF HUMAN IMMUNOGLOBULINS TO rzsI CEA BY RADIOIMMUNOELECTROPHORESIS Binding

Patient

diagnosis

Blood donors Prenatal Ca colon (local) Ca colon (disseminated) Other gastrointestinal Ca Other Ca Nonmalignant diseases

Number of patients sera tested

Nil

13 22 15 5 4 27 12

8 21 9 4 2 20 7

AS DETECTED

of lzsI CEAD I&

I@

U Some sera gave binding of lz51 CEA to IgG b Percentage of sera positive reflects number

weak

marked

1 1 0 0 0 2 0 and IgM. with binding

weak

3 0 3 1 1 0 4

marked

1 0 2 1 0 2 1

to either

4 0 3 0 1 4 4

or both

IgM

Percentb sera with Ig binding 38 5 40 20 50 26 42

and IgG.

342

MACSWEEN,

WARNER

AND

MACKAY

of human sera with addition of “‘1 FIG. 1. Radioimmunoelectrophoresis shows autoradiographs of IE patterns; lower half shows unstaine figure globulin serum and electrophoresis with cathl polyva lent rabbit anti-human with (a) chronic pancreatitis, (1~) diverticulit sera in wells were from patients colitis.

Percent Ammonium Patient Prenatal Ca colon

diagnosis

No inhibitor 27

(local)

Ca stomach Gastrointestinal hemorrhage ” Percent ‘9 was present as ’ Percent “‘1 c Qualitative phoresis.

34 22 25 33

sulfate

precipitation

of lJ”I

13 22 15

x 16

Rabbit

Upper half of pattern using right. Human (c) ulcerative

CEA

precipitation” 5 ng CEA

CEA. ,d IE ode to is, and

anti-HGG” 10 20 5 20 14

Binding in radioimmunoelectrophoresis’ NIL weak IgG NIL IgG IN

CEA precipitated after addition of ammonium sulfate. In one group 5 ng of CEA inhibitor. CEA precipitated after addition of rabbit anti-human gamma globulin, estimate of binding of ‘T CEA to immunoglobulin in radioimmunoeleetro-

CARCINOEMBRYONIC

ANTIGEN

IN WHOLE

SERUM

343

by Thompson et al. (4), ammonium sulfate was used to precipitate the complex of CEA with anti-CEA and the inhibitor used was perchloric acid extracts of the test human serum. With this method, it was found that positive assays occurred in a high proportion of sera from patients with cancer of the gastrointestinal tract although subsequent studies have shown that extracted sera from patients with some other types of cancer also react in the assay (Il,I2), as well as sera from some patients with nonmalignant disease (9). In considering the modifications in our assay system, it is therefore necessary to compare these results using whole serum with studies using extracted sera, and then to determine the specificity of our modified test for the patient groups studied. In scaling down the volumes of reagents used in the test so that only small amounts of patients serum were to be used, sufficient sensitivity of the assay system must be retained. When purified CEA is used as inhibitor, approximately 3 nglml of CEA can be detected when ammonium sulfate is used to precipitate the complex. Certain problems were encountered when whole serum (instead of PCA extracted serum) was introduced into the assay mixture. Some lzsI CEA was found to bind to some human globulins in amounts sufficient to mask the inhibition of binding to anti-CEA by the actual CEA in the whole serum, so that spuriously low values of CEA levels were recorded; this was overcome by the use of anti-serum to goat gamma globulin which specifically precipitates only the goat antibodies and does not precipitate complexes of lZ51-CEA with human globulin. In comparing two different sources of antiglobulin, it was found that with sheep antiserum, many blood donor sera gave high values, and, accordingly, all further tests used rabbit antisera to goat gamma globulin, which gave this result much less frequently. The reason for the false positive results using sheep antiserum could indicate that the presence of a globulin in some human sera inhibits the binding of sheep antibody to goat gamma globulin, i.e., human gamma globulin may antigenically cross react with goat gamma globulin. Alternatively there may be a natural antibody in human serum to sheep gammaglobulin. Our assay system with rabbit antiglobulins is very similar to that described by Egan et al. (Is), who have incorporated into the test several other technical controls; other aspects of the assay methodology are discussed by Warner and MacSween (manuscript in preparation). Four conclusions may be drawn from our results with the direct double antibody serum test on 376 human sera. (i) Most sera from local or disseminated colon cancer, from other gastrointestinal cancer, (chiefly stomach or pancreas), and from lung or breast, have CEA values higher than 90% of healthy persons. These results correspond to those described by other groups using PCA extracted sera, and in a preliminary report, with another test using unextracted serum (15). In our own series, most of the sera from patients with these carcinomas remained positive after extraction with perchloric acid further confirming the validity of results on tests on whole serum. (ii) Elevated CEA 1evels in serum are not found in all types of cancer, e.g.,

344

MACSWEEN,

WARNER

AND MACKAY

in our studies with leukemias, myeloma, cancer of the bladder, and certain other cancers, Many more tests must be performed before it can be stated-that CEA levels are not elevated in any given type of cancer. Indeed a few myeloma sera were positive in a series reported by Reynoso et al. (11). (iii) In considering sera from patients with nonmalignant disease, it is quite evident that this group contains more cases with elevated CEA levels over 5 nglml than the blood donor controls. Approximately 30-40% had levels over 5 rig/ml although only 10% had levels over 15 rig/ml, excepting pancreatitis and alcoholic cirrhosis. This forms a middle group between “normal” and “cancer” groups at each end. A similar result on unextracted sera from patients with inflammatory diseases of abdominal origin (chronic active hepatitis and ulcerative colitis) was reported (16) in that 30-35% of sera were positive for CEA (~2 nglml). However, when we extracted these groups of sera with PCA, a considerable reduction in incidence of positive tests was found, in that only approximately 10% had levels over 3 rig/ml. It would therefore appear that on direct serum tests, false positives can occur, meaning positive in tests on whole serum but negative after extraction with perchloric acid. (iv) Sera from patients with either alcoholic cirrhosis or pancreatitis behave in the CEA test like sera from gastrointestinal cancer rather than like the sera from other nonmalignant diseases. Thus, levels of over 15 rig/ml were frequently found (13/24), and most sera were still positive after extraction with PCA. These results are similar to those of Zamcheck (9). Several approaches to the elimination of false positive results were attempted. In some cases, pretreatment of the patients’ serum with normal goat serum eliminated the false positive results, and in two cases this was proven to be due to the presence in the patients’ serum of a “natural,” presumably crossreactive, antibody to goat gamma globulin which would inactivate the goat antibody to CEA. A more effective procedure in reducing the number of false positive assays was heating serum to about 90°C which aggregated most of the serum proteins. This may have removed from the assay inhibiting proteins, while CEA retained its antigenicity and remained in the supemate. This procedure would not limit assays on large numbers of sera. Further studies on this approach are in progress. The reason that occasionally patients with colon cancer lack detectable CEA in the serum could be that these patients have produced circulating antibody to CEA. In 1967 Gold (14) reported the presence of antibodies to CEA in such patients by hemagglutination, However, Collatz et al. (17) showed that this type of antibody is probably reactive against normal tissue proteins which can contaminate perchloric acid extracts of colonic tumors. Collatz et al. (17) found no evidence for antibodies to cancer specific antigen. Using radioimmunoassays, we failed to find definite evidence of antibodies to CEA. Many sera of patients having a wide range of diseases gave some degree of binding to ln51 CEA, as was also found with sera from some blood donors. In many cases, although a globulin was involved in the binding activity, inhibition of binding by a large excess of the unlabeled CEA did not occur; it appears that there may be a preferential binding of a nonspecific type, to the

CARCINOEMBRYONIC

ANTIGEN

IN

WHOLE

SERUM

345

iodinated antigen. Using radioimmunoelectrophoresis it could be shown that frequently IgM and/or IgG were responsible for this binding activity, although in only three sera (Table 13) was evidence of binding observed in all the assays used. While there is a suggestion of antibody activity in these three sera, the incomplete nature of the binding and inhibition precludes the definite demonstration of anti-CEA antibody. In most cases lz51 CEA binds in a nonspecific manner to immunoglobulin molecules. Our results suggest that a microradioimmunoassay can best be used to test small quantities of unextracted sera for the presence of CEA. Before the test can be used as a routine screening assay, the elimination of “false positives” is desirable. Even then, it appears that some nonmalignant diseases such as alcoholic cirrhosis may still be positive. The questions that must still be asked relate to whether the carcinoembryonic antigens that are found in low quantities in some normal or nonmalignant sera, in nonmalignant liver and gastrointestinal disease, and in the several different types of cancer, are one and the same, or whether there is a group of proteins that are antigenically related but are distinctive to different tissues. Prospective studies and a more comprehensive comparison of the direct serum test with the test on extracted sera now in progress will indicate whether the direct test and the test on extracted sera will give comparable results. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

GOLD, P. AND FREEDMAN, S. O.,J. Exp. Med. 121,439, 1965. GOLD, P. AND FREEDMAN, S. O.,j. Exp. Med. 122,467, 1965. KRUPEY, J., GOLD, P. AND FREEDMAN, S. O.,J. Exp. Med. 128,387, 1968. THOMPSON, D., KRUPEY, J., FREEDMAN, S. 0. AND GOLD, P., Proc. Nat. Acad. Sci. US, 64,161, 1969. MOORE, T. L., KUPCHIK, H. Z., MARCON, N. AND ZAMCHECK, N., Amer. J. Digest. Discuses, 16, 1, 1971. Lo GERFO, P., KRUPEY, J. AND HANSEN, H., N. Engl. J. Med. 285, 138, 1971. DHAR, P., MOORE, T. L., KUPCHIK, H. AND ZAMCHECK, N., Gastroenterology 69,655, 1971. MOORE, T. L., KUPCHIK, H. Z., DHAR, P. AND ZAMCHECK, N.,Gastroenterology 69,700,1971. ZAMCHECK, N., MOORE, T. L., DHAR, P. AND KUPCHIK, H., N. Engl.]. Med. 286,83, 1972. Lo GERFO, P., Lo GERFO, F., HERTER, F., BARKER, H. G. AND HANSEN, H., Amer. J. Surg. 123, 127, 1972. REYNOSO, G., CHU, T. M., HOLYOKE, D., COHEN, E., NEMOTO, T., WANG, J-J, CHUANC;, J., GURNAN, P. AND MURPHY, G. P.,J.A.M.A 220,361, 1972. National Cancer Institute of Canada and American Cancer Society, Can. Med. Ass. J. 107,25, 1972. GREENWOOD, F. C., HUNTER, W. M. AND GLOVER, J. S., Biochem. J. 89, 114, 1963. GOLD, P., Cancer 20, 1663, 1967. EGAN, M. L., LAUTENSCHLEGER, J. T., COLIGAN, J. E. AND TODD, C. W., Immunochemistry 9, 289, 1972. Go, V. L. W., SCHUTT, A. J., MOESTEL, C. G., SUMMERSKILL, W. H. J. AND Burr, H. R., Gastroenterology (in press). COLLATZ, E., VONKLEIST, S. AND BURTIN, P., Int. J. Cancer 8,298, 1971.