Improved performance of a double antibody radioimmunoassay for carcinoembryonic antigen

Journal of Immunological Methods, 25 ( 1 9 7 9 ) 3 1 1 - - 3 2 1

311

Q E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

IMPROVED PERFORMANCE OF A DOUBLE ANTIBODY R A D I O I M M U N O A S S A Y FOR CARCINOEMBRYONIC ANTIGEN

ROLF ZIMMERMAN

Department of Virology, National Bacteriological Laboratory, S-105 21 Stockholm, Sweden ( R e c e i v e d 12 J u n e 1 9 7 8 , a c c e p t e d 10 A u g u s t 1 9 7 8 )

A n e w d o u b l e a n t i b o d y solid-phase r a d i o i m m u n o a s s a y ( R I A ) for c a r c i n o e m b r y o n i c a n t i g e n ( C E A ) is critically a n a l y z e d . T h e aim o f t h e s t u d y was 4-fold: (a) t o d e f i n e t h e level o f sensitivity (a c o m p a r i s o n o f 3 d i f f e r e n t assay p r o c e d u r e s revealed t h a t o u r s e q u e n tial assay was m o r e sensitive t h a n m o s t previously r e p o r t e d RIAs, while c o m p e t i t i v e a n d n o n - e q u i l i b r i u m assay h a d wider m e a s u r i n g ranges); (b) to a n a l y z e recoveries o f C E A in e i t h e r s e r u m , p l a s m a or u r i n e ( t h e r e c o v e r y , even in urine, was very close t o e x p e c t e d values, i n d i c a t i n g t h a t n o CEA is lost or d e g r a d e d d u r i n g b r i e f storage o r in t h e extract i o n p r o c e d u r e ) ; (e) to e v a l u a t e inter- a n d intra-assay variations, since m o s t clinical m a n a g e m e n t is d e p e n d e n t o n serial assays r a t h e r t h a n single d e t e r m i n a t i o n s . T h e coefficients of v a r i a t i o n were low b o t h w i t h i n a n d b e t w e e n assays. A c h a n g e o f 3 ng C E A is r e q u i r e d for significant c h a n g e (> 2 S.D.) at t h e n o r m a l s e r u m level w h i c h is 16 ng C E A / ml in o u r assay. A t levels a b o v e n o r m a l , a c h a n g e o f 4 ng is r e q u i r e d ; (d) t h e assay was also d e v e l o p e d for d e t e r m i n a t i o n o f C E A levels in a large series o f p e r c h l o r i d acid t r e a t e d s e r u m , p l a s m a o r u r i n e samples. This f o r m s t h e basis for an assay s u i t a b l e for serial assays w i t h high sensitivity a n d a c c u r a c y in various n e o p l a s t i c diseases.

INTRODUCTION

A carcinoembryonic antigen (CEA) of the endodermal germ layer of the fetus has been described (Gold and Freedman, 1965). Amniotic fluid contains CEA throughout gestation (Gadler et al., 1978). Adenocarcinomas of the gastrointestinal tract contain high amounts of CEA (Gold and Freedman, 1965; Krupey et al., 1968) and small amounts are found in the normal gut (Martin and Martin, 1972). CEA can also be detected by sensitive methods in the serum of patients with gastrointestinal and other cancers (Thomson et al., 1969; Hansen et al., 1974; Rieger and Wahren, 1975; Zamcheck et al., 1975). CEA is a glycoprotein with a molecular weight of 180,000 daltons and has been investigated extensively for biochemical and immunological properties (Krupey et al., 1968; Hammarstr5m et al., 1976; for a recent summary, see Egan et al., 1977) and for clinical usefulness (Thomson et al., 1969; Hansen et al., 1974; Zamcheck et al., 1975). Several methods for quantitating CEA in b o d y fluids or tissues have been reported. Those used most frequently are radioimmunoassays: a modified Farr technique (Thorn-

312 son et al., 1969), the z-gel technique (Hansen et al., 1971), solid-phase or other double antibody techniques (Egan et al., 1972; Laurence et al., 1972; MacSween et al., 1972; Franchimont et al., 1974; Das et al., 1976; Frackelton et al., 1976; Kim et al., 1978). Other techniques used are passive hemagglutination inhibition (Anthony and Sosnowski, 1975), a m e t h o d using a gold-indium covered slide for visual detection of CEA (Giaever, 1976) and a solid-phase radioimmunoelectrophoresis (Saravis et al., 1974). A specific and sensitive assay to detect CEA in serum, plasma and urine is described here and critically analyzed. The most adequate parameters for controlling the quality of a radioimmunoassay are used (Rodbard et al., 1968; Siddiqui and Craig, 1975): (a) the 50% intercept as an index of sensitivity; (b) b o u n d (B)/total (T) labeled tracer, reflecting the immunoreactivity of the labeled antigen; (c) within assay variations; and (d) between assay variations. MATERIAL AND METHODS

Antigens Carcinoembryonic antigen (CEA), isolated and purified from liver metastases of colo-reactal carinomas, was kindly provided by Prof. Bounameaux, Hoffmann-La Roche, Basle, Switzerland (HLR-CEA) and Dr. S. HammarstrSm, Department of Immunology, University of Stockholm, Sweden (CEA 45, CEA 47). Non-specific cross-reacting antigen (NCA) (Mach and Pusztaszeri, 1972; Von Kleist et al., 1972) purified from human spleen was kindly provided by Dr. S. HammarstrSm (HammarstrSm et al., 1978). Bile glycoprotein 1 (BGP1) isolated from human hepatic bile was kindly provided by Dr. T. Svenberg, Department of Immunology, University of Stockholm, Sweden. The purification procedure and the physico-chemical and immunological properties of this CEA cross-reacting antigen have been reported by Svenberg (1976). Labeling of antigens [12sI]CEA and [12SI]NCA were prepared by a modified chloramine-T m e t h o d (Hunter, 1971). 50 pg of CEA was labeled in a P10 vial containing 2 mCi [12SI]Na (Radiochemical Center, Amersham, England) adjusted to pH 8.4 with 0.5 M borate buffer, pH 8.4. Four t~g chloramine-T/~g protein in 0.05 M borate buffer were added. After mixing the reagents for 60 sec, the unreacted iodine was reduced back to iodide with sodium metabisulfite, 8 ~g/pg protein. Unreacted 12sI was removed on a Sephadex G-50 fine column (Pharmacia, Uppsala, Sweden), equilibrated with 0.1 M Tris-HC1 buffer, pH 7.5, containing 1% bovine serum albumin (BSA) and 0.02% Na azide. The serum albumin serves to saturate the absorption sites on the gel, thus diminishing non-specific binding to gel and glassware. The eluate was collected in 1 ml fractions and 25 ~l counted in a 7-counter (Packard Auto-Gamma Model 5110 Scintillation spectrometer). 1 - - 3 m l from the void peak with the highest radioactivity were used as tracer. Usu-

313 ally 50% labeling efficiency was obtained, corresponding to a specific activity of 20--30 pCi/pg protein. When kept at +4°C the labeled tracer could be used for 1--2 months w i t h o u t much loss of antibody binding activity. A n tisera

Anti-CEA antisera were produced in rabbits by two intracutaneous injections of 50 pg purified CEA separated by a week's interval, followed by an intramuscular booster of 100 pg CEA in Freund's complete adjuvant. The rabbits were bled weekly, and reactive sera pooled and absorbed. Absorption was carried o u t once with packed human A and B red blood cells, 3 times with glutaraldehyde insolubilized immunosorbents (Avrameas and Ternynck, 1969) of human serum and perchloric acid (PCA) treated water extract from normal human lung, containing NCA. Rabbit anti-NCA was produced in a similar way and kindly provided b y Dr. S. HammarstrSm. This antiserum is highly specific to NCA; 5 ng of NCA gives 50% inhibition b u t > 1 pg of CEA does n o t inhibit the NCA-anti-NCA reaction (Gadler et al., 1978; HammarstrSm et al., 1978). A n t i s e r u m t i t r a t i o n . 100 gl of antiserum dilutions ranging from 1 : 100 to 1:32000 were mixed with 1 0 0 p l of 12SI-labeled tracer and 400 ~1 phosphate-buffered saline (PBS) containing 1% BSA. Incubation was carried o u t for 16 h at room temperature. The absorbed CEA-specific antiserum used in this study had an initial titer of 3000 (Fig. 1). A final antiserum dilu80-

60 a~

\ 40 .D g.I

\

\

\

\

\

0

20

1:100

1:500

1:2 000

1:32 0 0 0

1:8 0 0 0

anti-CEA -antiserum

dilution

Fig. 1. A n t i s e r u m t i t r a t i o n curve. 12SI-labeled CEA b o u n d (B) by a b s o r b e d rabbit antiC E A e x p r e s s e d as a p e r c e n t a g e o f t o t a l (T) [125I]CEA a d d e d .

314 tion of 1 : 18,000 binding 65% of m a x i m u m precipitable [12sI]CEA tracer, was used.

Serum, plasma and urine samples Patient plasma samples were collected in Vacutainer tubes (BectonDickinson, Rutherford, NJ) containing liquid ethylenediaminetetraacetate (EDTA). After centrifugation within 6 h of collection, the plasma was separated. Blood for serum was collected in Vacutainer tubes without additives. After clotting at room temperature for 2 h, serum was collected after centrifugation. Plasmas and sera were stored at --20°C. Normal serum samples were obtained from 174 healthy women, constituting a control group in a clinical study (Rimsten et al., 1978). Urine samples were taken from healthy persons. All specimens were obtained aseptically. Extractions were performed by adding 0.5 ml of 2 M perchloric acid (PCA) to 0.5 ml serum, plasma or urine. After centrifugation at 3000 × g for 30 min, the PCA-soluble supernatant was transferred to dialysis tubings (Union Carbide Corp., Chicago, IL). Dialysis was carried out for 6 h in running tap water, in distilled water overnight and in PBS for 3 h.

Radioimmunoassay procedures 200 gl of PCA-extracted serum, plasma or urine samples or predetermined CEA amounts were mixed with 100 ~1 of anti-CEA antiserum diluted in PBS containing 1% BSA and incubated at 37°C in 15 mm × 100 mm polystyrene tubes (Nunc A/S, Roskilde, Denmark). After the first incubation, 100 ~1 of [12sI]CEA tracer (about 2 ng and l 0 s counts/min) and 200 pl of PBS were added. Different incubation procedures. Standard inhibition curves were made using 3 different procedures. (a) In the competitive assay, the anti-CEA antiserum was incubated for 16 h at room temperature together with standard unlabeled CEA and the ~2SI-labeled CEA tracer. (b) In the sequential procedure, the antiserum and standard CEA were incubated for 6 h at 37°C prior to addition of [12sI]CEA. This mixture was then incubated for 16 h at room temperature. (c) A non-equilibrium assay, similar to the commercial kit procedure of Hoffmann-La Roche, was also performed. Antiserum and standards were incubated for 30 min at 45°C and with [12sI] CEA for another 30 min at 45°C. The separation of antigen-antibody complexes from free labeled antigen was performed utilizing the DASP suspension as below. Double antibody solid phase (DASP). To separate free antigen from antigen bound to the specific rabbit antibody, an immunoadsorbent second antibody was employed. Goat anti-rabbit antibodies are chemically bound to cellulose beads DASP (Campbell and Weliky, 1967) purchased from Organon, Oss, The Netherlands. One ml of a 1 : 10 dilution of the DASP suspension in PBS was added to each tube. Incubation for 2 h at 37°C with interm i t t e n t agitation was required to precipitate m a x i m u m rabbit anti-CEA. Antigen-antibody complexes were sedimented at 1000 × g for 5--10 min.

315

The supernatants were removed and after one wash with 1 ml PBS the pellets were counted. All values are means of double or triple determinations.

Analysis of data The data were analyzed on-line with a Compucord 327 Scientist desk-top computer and a tape cassette drive, model 392 (Compucorp, Los Angeles, CA). The standard curve was divided into a left and a right hand curve, with the middle concentration of the standard curve c o m m o n to both. A thirddegree curve was fitted to each of the curves. The antigen concentration in an u n k n o w n sample was calculated by solving either of the third-degree equations. In our experience, this curve-fitting m e t h o d works very well with several commercial RIA kits, and fits a manually plotted standard curve better than a slope calculated by linear regression. RESULTS

The maximal percentage of [12sI]CEA precipitated by anti-CEA antiserum in the absence of unlabeled antigen (% b o u n d / t o t a l counts/min) was 60--80%, reflecting a high immunoreactivity after labeling of the antigen (Fig. 1). The percentage bound at the final working antiserum dilution of

3o! Ira

C D 0

"20 < Z

,°l1

anti~

NCA

anti-CEA

I: 500

1:iO00

1 :8000 antiserum

dilution

Fig. 2. C o m p a r i s o n o f t h e ability o f a n t i - C E A a n d a n t i - N C A a n t i s e r u m to p r e c i p i t a t e 12SI-labeled NCA. • $, a n t i - C E A ; o ©, anti-NCA.

316

1 : 18,000 was always in the range of 38--52%, depending on the time after labeling of the antigen. ~2SI-labeled CEA tracer added in amounts from 0.2 to 12 ng did n o t change the shape of the antibody titration curve. This indicates optimal immunoreactivity of the tracer in the concentrations used. Fig. 2 shows that the anti-CEA antiserum does not react significantly with 12SI-labeled NCA tracer. In the working antiserum dilution, only 2% of the total a m o u n t of labeled NCA tracer was precipitated. One per cent of either tracer was precipitated in the absence of the first antibody, this is referred to as non-specific binding. The specificity of the anti-CEA antiserum was also shown in standard curves and hybrid standard curves (Fig. 3). 1,5 ng CEA, more than 6 ~g NCA and 200 ng BGP I were required to give 50% inhibition with the absorbed anti-CEA antiserum.

Comparison of sequential and competitive procedures The different assay procedures are compared in Fig. 4. The standard inhibition curves were performed using the same dilution of anti-CEA antiserum, the same amounts of standard CEA and equal amounts of DASP suspension. When using the 50% intercept as an index of sensitivity, the sequential procedure requires 1.3 ng CEA ranging from 1.15 to 1.55 ng; the

100

8O

6O

m 40

NCA

20

~

-~

CEA 45 C E A 47 HLR-CEA

0,1

1.0

10

100

1000

10,000 ng

Fig. 3. C o m p a r i s o n o f s t a n d a r d i n h i b i t i o n curves i n h i b i t e d b y C E A p r e p a r a t i o n s a n d h y b r i d s t a n d a r d curves i n h i b i t e d b y cross-reacting antigens. T h e a m o u n t o f [ 1 2 s I ] C E A is expressed as a p e r c e n t a g e o f m a x i m a l [ 1 2 s I ] C E A b o u n d in t h e a b s e n c e of u n l a b e l e d CEA (% B/B0). B0 = m a x i m a l % b o u n d [ 1 2 s I ] C E A at t h e a n t i s e r u m d i l u t i o n used. $ ~, NCA; &-&,BGP 1;" -,CEA 45;© ©, C E A 4 7 ; D ~, H L R - C E A .

317

100

80

6o

.Q <

40

20

0.'19

0.'38

0.78

1:56

3112

6,'25

12.5

2'5.0

ng CEA Fig. 4. Comparison of standard inhibition curves by the different incubation procedures. The amount of [12SI]CEA bound in per cent of maximal [12sI]CEA bound in the absence of unlabeled CEA (% B/B0). B0 = maximal % bound [12SI]CEA at the antiserum dilution used. ; l , sequential assay for 6 h at 37°C and 16 h at 20°C; © ©, competitive assay for 16 h at 20°C; • • , non-equilibrium competitive assay for 30 min at 45°C and 30 rain at 45°C.

competitive and the non-equilibrium methods require 3.8 ng CEA. The sequential procedure has a steeper dose-response curve and the least detectable dose giving a significant inhibition (>10%) was 0.25 ng CEA. When the other two procedures were used, the assays showed wider measuring ranges but were less sensitive and the least detectable dose was 0.50 ng CEA.

Recovery o f CEA in PCA-extracted samples To aliquots of 4 human plasma pools, containing 5.8, 6.0, 6.5 or 10.2 ng CEA/ml respectively, and to 3 pools of urine, containing 18.4, 21.0 or 29.0 ng CEA/ml respectively, known amounts of standard CEA were added. The samples were then treated as an u n k n o w n with PCA extraction and dialysis. The CEA values obtained were compared to expected values. Table 1 shows the results with one plasma and one urine pool. The CEA recovery for all serum and plasma experiments showed a range of 83--102%, average 94%. For urine the range was 87--115%, average 106%. When+the expected and actual CEA values were compared by linear regression, the correlation

318

TABLE 1 RECOVERY OF CEA WITH A SEQUENTIAL RADIOIMMUNOASSAY ng CEA in

ng CEA

Plasma

Added standard

Expected

Obtained

6.5 6.5 6.5 6.5 Urine 29.0 29.0 29.0

1.6 3.1 6.3 12.5

8.1 9.6 12.8 19.0

7.6 8.7 12.0 17.9

94 91 94 94

3.1 12.5 25.0

32.1 41.5 54.0

33.0 48.0 57.2

103 115 105

%recovery

c o e f f i c i e n t s f o r all m a t e r i a l s w e r e r = 0 . 9 7 1 0 - - 0 . 9 9 9 5 . This indicates t h a t C E A was n o t m e a s u r a b l y c h a n g e d in p l a s m a , s e r u m o r urine d u r i n g t h e PCA extraction procedure. In o r d e r t o c o n t r o l h o w t h e PCA e x t r a c t i o n a n d dialysis t r e a t m e n t a f f e c t t h e a c c u r a c y o f t h e assay t h e f o l l o w i n g e x p e r i m e n t was d o n e : t w e n t y 0.5 m l samples from a pool of normal human serum each containing l0 s counts/min [12sI]CEA w e r e PCA e x t r a c t e d a n d d i a l y s e d ; 2 0 0 ul s a m p l e s in d u p l i c a t e s w e r e t h e n d r a w n f r o m t h e t o t a l v o l u m e r e c o v e r e d f r o m e a c h dialysis t u b i n g . T h e c o e f f i c i e n t o f v a r i a t i o n o f t h e c o u n t s / m i n r e c o v e r e d was 6%.

Assay variations T h e a c c u r a c y o f t h e s e q u e n t i a l R I A in t e r m s o f within- and b e t w e e n - a s s a y v a r i a t i o n is s h o w n in T a b l e 2. T h e s a m p l e s w e r e r u n with or w i t h o u t p r i o r PCA e x t r a c t i o n . T h e w i t h i n - a s s a y v a r i a t i o n was 1 - - 2 . 5 % f o r P C A - e x t r a c t e d as well as f o r n o n - P C A - e x t r a c t e d p l a s m a pools. T h e n o n - P C A - e x t r a c t e d p l a s m a s a m p l e s h a d 2--15% higher C E A values t h a n a f t e r P C A e x t r a c t i o n . W h e n assaying urines, 3 6 - - 9 2 % o f t h e m e a s u r a b l e value in n o n - P C A - e x t r a c t e d samples r e m a i n e d a f t e r P C A t r e a t m e n t . An e s t i m a t i o n o f t h e b e t w e e n - a s s a y v a r i a t i o n was m a d e w i t h PCA- and n o n - P C A - e x t r a c t e d s a m p l e s f r o m p o o l s o f n o r m a l p l a s m a , s e r u m and urine. T h e b e t w e e n - a s s a y v a r i a t i o n o f s e r u m and p l a s m a pools, used at intervals d u r i n g 1 y e a r , was f o u n d t o be 16.5% f o r P C A - e x t r a c t e d s e r u m a n d 13.4% for PCA-extracted plasma whereas the non-extracted serum and plasma both h a d a v a r i a t i o n o f 8.2%. T h e c o e f f i c i e n t o f v a r i a t i o n f o r P C A - e x t r a c t e d urine was 20%. T h e m e a n s e r u m C E A values o f 174 h e a l t h y w o m e n was 10.5 _+ 2.3 n g / m l . In T a b l e 2 these c o n t r o l s are divided i n t o 3 groups: 1 5 4 s a m p l e s w i t h C E A values less t h a n 13 n g / m l h a d a m e a n a n d S.D. o f 9.9 _+ 1.4; 20 s a m p l e s w i t h a C E A o f > 1 3 n g / m l h a d a slightly higher S.D. o f 1.7.

319 TABLE 2 VARIATIONS OF THE SEQUENTIAL CEA RADIOIMMUNOASSAY Plasma/serum lot no.

No. of comparisons

ng CEA -+ S.D. PCA extraction

No PCA treatment

10 10 10 10

5.8±0.13 6.0±0.13 6.5±0.10 10.2±0.11

6.1±0.07 6.1±0.10 6.7±0.14 11.8±0.23

12 18 8

8.6 -+ 1.42 10.7 -+ 1.43 5.5 ± 1.10

10.0 -+ 0.82 10.0 ± 0.82 not done

Within- assay variation

Plasma Plasma Plasma Plasma

1 2 3 4

Between-assay variation

Plasma 5 Serum 6 Urine 7 N o r m a l serum controls

CEA 0--13 ng >13 >16

154 16 4

9.9 +- 1.43 14.4 -+ 1.75 18.8 -+ 1.76

DISCUSSION T h e p r e s e n t s t u d y was p r i m a r i l y designed t o e v a l u a t e t h e s p e c i f i c i t y and sensitivity o f a d o u b l e a n t i b o d y C E A R I A . No p a t i e n t d a t a are p r e s e n t e d h e r e , b u t p r e v i o u s studies h a v e s h o w n t h a t t h e a c c u r a c y o f o u r assay in d e t e c t i n g raised C E A levels in t u m o r p a t i e n t s is similar t o t h a t o f t h e z-gel assay o f H o f f m a n n - L a R o c h e (Rieger and Wahren, 1 9 7 5 ; W a h r e n et al., 1975). In o u r s e q u e n t i a l assay t h e 50% i n t e r c e p t falls b e t w e e n 1.15 a n d 1.55 ng CEA, w h i c h d e m o n s t r a t e s t h a t it is a m o r e sensitive assay t h a n m o s t p u b lished or c o m m e r c i a l R I A s f o r CEA. T h e i m m u n o r e a c t i v i t y o f t h e labeled a n t i g e n was high and t h e assay v a r i a t i o n s w e r e low. A n t i s e r u m s p e c i f i c i t y is crucial f o r t h e reliability o f values o b t a i n e d f r o m r a d i o i m m u n o a s s a y s . T h e a n t i - C E A a n t i s e r u m u s e d in this s t u d y was m a d e CEA-specific b y a b s o r b i n g o u t a n t i b o d y m o l e c u l e s r e a c t i n g w i t h N C A a n d b l o o d g r o u p s u b s t a n c e s . N o c o r r e c t i o n f o r n o r m a l s e r u m C E A values, E D T A or o t h e r additives to t h e s t a n d a r d i n h i b i t i o n c u r v e w e r e m a d e , in c o n t r a s t t o w h a t has b e e n d o n e p r e v i o u s l y ( W a h r e n et al., 1 9 7 5 ) , a n d t h e s t a n d a r d c u r v e s a m p l e s did n o t c o n t a i n E D T A , as t h e y d o in o t h e r assays ( H a n s e n et al., 1971). I t is t h u s p r e s u m e d t h a t a c c u r a t e t o t a l C E A s e r u m o r p l a s m a values are m e a s u r e d w i t h t h e p r e s e n t m e t h o d . As far as t h e assay p r o c e d u r e s are c o n c e r n e d , t h e s e q u e n t i a l assay has t h e s t e e p e s t d o s e - r e s p o n s e c u r v e a n d is t h u s m o r e sensitive. O n t h e o t h e r h a n d t h e c o m p e t i t i v e a n d n o n - e q u i l i b r i u m assays h a v e a w i d e r m e a s u r i n g

320

range. It is therefore possible to use the competitive assays for screening, while quantification in the range of 0.1--1 ng is more reliable with the sequential assay. The measuring range of the sequential assay for clinical purposes is adequately wide. In general, therefore, specimens do not have to be diluted. A requirement for the assay was also to conveniently process large numbers of samples. The assay has been used to measure serum and urinary CEA in clinical material (Wahren et al., 1975; Rimsten et al., 1978). 97% of healthy women with a mean age of 63 years had values below 16 ng CEA/ml. The CEA mean and S.D. values in these samples were 10.5 ± 2.3 ng/ml. Therefore our upper normal serum or plasma limit has been set at 16 ng CEA/ml. The PCA extraction and dialysis treatment affect the accuracy of the assay very little. Reproducible volumes were recovered from the dialysis tubings and the samples contain reproducible amounts of CEA. Our assay usually includes the perchloric acid extraction step with subsequent dialysis, although we found only small differences between PCA-extracted and nonPCA-extracted samples of serum and plasma. With urine, however, PCA treatment seems mandatory. PCA treatment precipitates urinary material inhibitory in the CEA assay, the nature of which is not yet precisely known (Nery et al., 1974). Some of this may be CEA, although our r e s u l t s - showing complete recovery of standard CEA in urine -- make this less likely. Other authors have found little or nor CEA in the PCA precipitates of plasma (Sorokin et al., 1973). In randomly collected urinary specimens from patients with bladder cancer (Hall et al., 1972; Wahren et al., 1975) increased CEA levels as compared with healthy persons have been reported, and verified with 24 h urines (Ionescu et al., 1976; and our own observations). The increased levels found in the urine of patients with bladder cancer are derived at least partly from the t u m o r cells. This has been demonstrated in exfoliated t u m o r cells and in t u m o r tissue (Wahren et al., 1977; Zimmerman and Hammarstr5m, 1978). Serum or plasma CEA from healthy persons occurs in the range 1--20 ng/ ml for all methods used at present. With malignant disease, an increased value probably reflects circulating t u m o r substances, since the value decreases with successfully treated tumors (Tomson et al., 1969; Hansen et al., 1974; Rieger and Wahren, 1975). Monitoring of patients with malignant disease requires fast, specific and sensitive radioimmunoassays similar to the one described here. REFERENCES Anthony, R.L. and K.M. Sosnowski, 1975, Clin. Immunol. Immunopathol. 4 , 3 6 2 . Avrameas, S. and T. Ternynck, 1969, Immunochemistry 6, 66. Campbell, D.H. and N. Weliky, 1967, in: Methods in Immunology and Immunochemistry, Vol. 1, eds. C.A. Williams and M.W. Chase (Academic Press, New York) p. 378.

321 Das, S., B.R. Das and M.D. Terry, 1976, Cancer Res. 36, 1961. Egan, M., J. Lautenschleger, J. Coligan and C. Todd, 1972, Immunochemistry 9 , 2 8 9 . Egan, M., E. Engvall, E. Rouslahti and C. Todd, 1977, Cancer 40, 458. Frackelton, Jr., A.R., R.P. Szaro and J.K. Weltman, 1976, Cancer Res. 36, 2845. Franchimont, P., M.L. Debruche, P.F. Zangerlee and J. Proyard, 1973, in: International Atomic Energy Agency Symp. on Radioimmunoassay and Related Procedures in Clinical Medicine and Research, Instanbul, p. 267. Gadler, H., K. Bremme, B. Wahren and S. HammarstrSm, 1978, Cancer 42, 1579. Giaever, I., 1976, J. Immunol. 116, 766. Gold, P. and S.O. Freedman, 1965, J. Exp. Med. 122,467. Hall, R.R., D.J.R. Laurence, D. Darcy, U. Stevens, R. James, S. Roberts and A.M. Neville, 1972, Br. Med. J. 3 , 6 0 9 . HammarstrSm, S., E. Engvall and G. Sundblad, 1976, in: Skandia International Symposia: Health Control in Detection of Cancer, eds. H. BostrSm, T. Larson and N. Ljungstedt (Almqvist and Wiksell, Stockholm) p. 24. HammarstrSm, S., T. Svenberg, A. Hedin and G. Sundblad, 1978, Scand. J. Immunol. 7 (Suppl. 6) 33. Hansen, H.J., K.P. Lance and J. Krupey, 1971, Clin. Res° 19,143. Hansen, H.S., J.J. Snyder, E. Miller et al., 1974, Hum. Pathol. 5 , 1 3 9 . Hunter, W., 1971, in: Radioimmunoassay Methods, eds. K.E. Kirkham and W.M. Hunter (Churchill-Livingstone, Edinburgh) p. 3 Ionescu, G., N.A. Romas, L. Ionescu, S. Bennett, M. Tannenbaum, R.J. Veenema and J.K. Lattimer, 1976, J. Urol. 115, 46. Kim, Y.D., J.T. Tomita and J.R. Schenck, 1978, J. Immunol. Methods 19, 309. Krupey, J., P. Gold and S.O. Freedman, 1968, J. Exp. Med. 128,387. Laurence, D.J.R., U. Stevens, R. Bettelheim, D. Darcy, C. Leese, C. Turberville, P. Alexander, E.W. Johns and A.M. Neville, 1972, Br. Med. J. 9 , 6 0 5 . Mach, J.P. and G. Pusztaszeri, 1972, Immunochemistry 9, 1031. MacSween, J.M., N.L. Warner, A.D. Bankhurst and I.R. MacKay 1972, Br. J. Cancer 26, 356. Martin, F. and M.S. Martin, 1972, Int. J. Cancer 9 , 6 4 1 . Nery, R.~R. James, A.L. Barsoum and H. Bullman, 1974, Br. J. Cancer 29,413. Rieger, ~ and B. Wahren, 1975, Scand. J. Gastroenterol. 10,869. Rimsten,/~., H.-O. Adami, B. Wahren and B. Nordin, 1978, in preparation. Rodbard, D., P.L. Rayford, J.A. Cooper and G.T. Ross, 1968, J. Clin. Endocr. 28, 1412. Saravis, C.A., N. Zamcheck, H.Z. Kupchik, R. McCabe and D. Munjal, 1974, J. Natl. Cancer Inst. 5 3 , 9 2 1 . Siddiqui, S.A. and A. Craig, 1975, Med. Lab. Techn. 32,171. Sorokin, J.J., H.Z. Kupchik and N. Zamcheck, 1973, J. Natl. Cancer Inst. 51, 1081. Svenberg, T., 1976, Int. J. Cancer 17,588. Thomson, D.M.P., J. Krupey, S.O. Freedman and P. Gold, 1969, Proc. Natl. Acad. Sci. 64,161. Von Kleist, S., G. Chavanel and P. Burtin, 1972, Proc. Natl. Acad. Sci. U.S.A. 69, 2492. Wahren, B., F. Edsmyr and R. Zimmerman, 1975, Cancer 36, 1490. Wahren, B., P. Esposti and R. Zimmerman, 1977, Cancer 40, 1511. Zamcheck, N., W.G. Doos, R. Prudente, B.B. Lurie and L.S. Gottlieb, 1975, Hum. Pathol. 6, 31. Zimmerman, R. and S. HammarstrSm, 1978, Urol. Res., in press.