A Monoclonal Antibody--Enzyme Immunoassay for Serum Carcinoembryonic Antigen withIncreased Specificity for CarcinomasAuthor(s): Anders Hedin, Leif Carlsson, Asta Berglund and Sten HammarstromSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 80, No. 11, [Part 1: Biological Sciences] (Jun. 1, 1983), pp. 3470-3474Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/14557 .

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Proc. Natl. Acad. Sci. USA Vol. 80, pp. 3470-3474, June 1983 Medical Sciences

A monoclonal antibody-enzyme immunoassay for serum carcinoembryonic antigen with increased specificity for carcinomas

(nonspecific crossreacting antigens 1 and 2/conventional radioimmunoassay)

ANDERS HEDIN*, LEIF CARLSSONt, ASTA BERGLUNDt, AND STEN HAMMARSTROM* *Department of Immunology, University of Stockholm, S-106 91, Stockhol, Sweden; and tPharmacia Diagnostics AB, Box 17, S-751 03, Uppsala, Sweden

Communicated by Werner Henle, February 3, 1983

ABSTRACT A two-site monoclonal antibody-enzyme immu- noassay (MEIA) for carcinoembryonic antigen (CEA) was devel- oped that uses two monoclonal anti-CEA antibodies, which rec- ognize two different epitopes in the peptide moiety of CEA. The assay was sensitive to 0.5 j/g/liter and had a measuring range of 0.5-200 ,/g of CEA per liter. It was highly specific inasmuch as none of three known CEA-related substances, "nonspecific cross- reacting antigens I and 2" (NCA-1 and NCA-2) and biliary gly- coprotein I (BGP I), reacted in the assay. NCA-2 (meconium an- tigen) is very similar to CEA. None of five commercially available CEA assays were able to differentiate between CEA and NCA-2. With one exception (colon), normal tissue extracts did not react in the MEIA even when tested at very high concentrations. Sera from a total of 180 healthy individuals and patients with malignant and nonmalignant disease were analyzed for CEA levels by using the MEIA and in parallel a conventional radioimmunoassay. A sig- nificant increase in specificity for carcinomas was obtained with the MEIA. This was essentially due to a decrease of MEIA CEA values in sera from patients with nonmalignant disease. The CEA values in the group of carcinoma patients (colon, pancreas, lung, and breast) were the same in the two assays.

Although existing carcinoembryonic antigen (CEA) assays can be used successfully for monitoring treatment of cancer and as an independent prognostic tool, they have limited value as an independent test to establish the diagnosis of cancer (1). An important goal in current research on CEA is therefore to im- prove the cancer specificity of the test. If tumor CEA is chem- ically different from the molecules responsible for CEA activity in the blood of healthy individuals and patients with various nonmalignant diseases, it should be possible to make mono- clonal antibodies against such "discriminating areas" in CEA. Such antibodies may then be used to construct CEA assays with higher disease-related specificity.

Three CEA-crossreactive normal tissue components have been identified so far. One of these, nonspecific crossreacting an- tigen 2 (NCA-2), present in relatively high concentrations in meconium (2, 3), is very closely related to tumor CEA. In fact, most conventional anti-CEA sera do not discriminate between these two macromolecules. We have recently produced a series of monoclonal antibodies against CEA, several of which were of high affinity and directed against different conformation-de- pendent epitopes of the CEA molecule (4).

In this communication we report the development of a two- site monoclonal antibody-enzyme immunoassay (MEIA) for CEA that shows an increased disease-related specificity for carci- nomas compared to a conventional radioimmunoassay (RIA).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U.S.C. ?1734 solely to indicate this fact.

MATERIALS AND METHODS Monoclonal Antibodies (MAbs). Four anti-CEA MAbs-9,

27, 38S1, and 46 (4)-were used in the study. They were all of the IgG1 K subclass and recognized three different epitopes in the peptide moiety of the CEA molecule; epitope A, MAb 9; epitope C, MAb 27; epitope D, MAb 38S1 and MAb 46 (4). Both epitope C and epitope D are present at least twice in the CEA molecule, whereas epitope A appears to be present only once (4). The affinity constants for the interaction between CEA and MAbs 9, 27, and 38S1 were 7.4 x 108, 3.3 x 108, and 1.2 X 108 M-1, respectively (4). The value for MAb 46 has not been determined. None of the antibodies reacted with NCA or bil- iary glycoprotein I (BGP I) (4).

Highly purified MAb was obtained by passage of ascitic fluid over an immunoadsorbent column consisting of CEA conju- gated to cyanogen bromide-activated Sepharose 4B (Pharmacia, Uppsala, Sweden). Antibodies were eluted with 6 M guani- dine-HC1.

CEA and Related Materials. CEA, low molecular weight CEA (CEAlow), and NCA (5, 6) were purified from individual liver metastases of colorectal cancer as described (7, 8, 9). The antigens were homogeneous on gel filtration and gave a single relatively broad band on sodium dodecyl sulfate/polyacryl- amide gel electrophoresis (7, 8, 9). Highly purified BGP I was prepared from hepatic bile of nonicteric patients as described (10).

NCA-2 was purified from pooled meconium (77 g wet weight) of nine infants essentially as described by Matsuoka et al. (3). Briefly, meconium extract was precipitated with 1.0 M perchlo- ric acid and the acid-soluble fraction was applied to a calf anti- CEA immunoadsorbent. After washing, semipurified NCA-2 was eluted from the adsorbent with 0.1 M glycine-HCl, pH 2.3. The eluate was then fractionated on a Sepharose 6B column. Most of the CEA-like activity was recovered in two peaks. The elution volume of the material in the first peak was similar to that of CEA. It did not react with specific anti-NCA serum and must therefore be NCA-2 as defined by Matsuoka et al. (3). The material in the second peak reacted with specific NCA serum. Purified NCA-2 was also obtained from P. Burtin (Villejuif, France) (2).

Highly purified orosomucoid (al acid glycoprotein) was a gift from H. P. Ekre (Kabi, Stockholm). The perchloric acid-soluble (1.0 M) fraction from the following human tissues or secretions was prepared: liver metastases of colorectal cancer (3 samples), urinary bladder carcinoma (1 sample), prostate carcinoma (1

Abbreviations: CEA, carcinoembryonic antigen; NCA, nonspecific crossreacting antigen; BGP, biliary glycoprotein; MEIA, monoclonal an- tibody-enzyme immunoassay; MAb, monoclonal antibody; RIA, ra- dioimmunoassay; ELISA, enzyme-linked immunoadsorbent assay.

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Proc. Natl. Acad. Sci. USA 80 (1983) 3471

sample), normal adult colon (1 sample, 5 cm from tumor), nor- mal adult spleen (pool of 20 spleens), normal adult lung (pool of 3 lungs), normal adult liver, muscle, bile, and feces (1 sample each), and meconium (pool from 9 individuals). The material was dialyzed and lyophilized. Saline extracts from normal hu- man spleen, liver, and muscle were also prepared.

Patients' Sera. Serum samples from 180 men and nonpreg- nant women were studied: 40 sera from healthy individuals (20 smokers and 20 nonsmokers), 45 sera from patients with non- malignant liver disease (cirrhosis associated with alcohol abuse, n = 27; chronic hepatitis, n = 12; Wilson disease, n = 4), 25 sera from ulcerative colitis patients, and 70 sera from patients with malignant disease. The diagnosis and number of cancer patients were as follows: colorectal carcinoma (n = 25, metas- tases in 13), pancreatic carcinoma (n = 15, metastases in 8), lung carcinoma (n = 15, metastases in 8), breast carcinoma (n = 5, metastases in 1), melanoma (n = 4, metastases in 2), sarcoma (n = 4, metastases in 1), lymphoma (n = 2). In the comparative analyses sera were diluted 1:5 in phosphate-buffered saline, pH 7.4, containing 5% porcine serum prior to assay.

MEIA for CEA. Systemic testing of different combinations of the four MAbs-9, 27, 38S1, and 46-showed that the most sensitive assay for CEA was obtained when MAbs 38S1 and 27 were used in combination. These antibodies recognize two dif- ferent epitopes (Fig. 2). MAb 38S1 in the form of unpurified ascitic fluid was coupled to the solid phase. Four-tenths mil- liliter of ascitic fluid containing approximately 0.5 mg of MAb was used per g of CNBr-activated paper discs (Pharmacia, Uppsala, Sweden). Coupling was performed in 0.1 M NaHCO3 at 4?C for 18 hr in rotating (150 rpm) test tubes. Unreactive groups were blocked with 1 M ethanolamine. The enzyme conjugate was prepared by coupling immunoadsorbent-purified MAb 27 to P-galactosidase from Escherichia coli (type VI, Sigma) via thiol groups as described by Carlsson et al. (11). Efficient con- jugation was obtained with an enzyme-to-MAb ratio of ap- proximately 2.

The MEIA was performed as follows: 100 A1 of serum (or standard CEA) was added to a test tube containing one MAb 38S1-coated paper disc. Then 50 ,ul of MAb 27-enzyme con- jugate (2.0 ,tg/ml in phosphate-buffered saline containing 3% bovine serum albumin, 1% normal mouse serum, and 0.5% Tween 20) was added and the reaction mixture was incubated with shaking (1,400 rpm) at 20?C overnight. After three wash- ings with saline, 200 1d of o-nitrophenyl-,3-D-galactopyranoside (2.1 mg/ml, Fluka AG, Buchs, Switzerland) was added. The tubes were then incubated at 37?C for 30-100 min and the re- action was stopped by the addition of 1.0 ml of 0.43 M Na2CO3. The amount of colored product was measured by absorbance at 420 nm.

Other CEA Immunoassays. Sera tested in MEIA were also assayed in parallel with a conventional sandwich RIA, Phadebas CEA PRIST (Pharmacia, Uppsala, Sweden). This assay uses specific sheep and rabbit antisera and has a lower limit of sen- sitivity of =1 Ag of CEA per liter. Sera were diluted 1:5 in phosphate-buffered saline plus 5% porcine serum before anal- ysis.

The influence of CEA-related normal tissue components, notably NCA-2, in five commercial CEA immunoassays was also studied. The following assays were investigated: CEA Roche Test Kit (Z-gel assay) and CEA EIA-Test (sandwich test using one MAb), both from Hoffmann-La Roche (Basel, Switzerland); Abbott CEA RIA Diagnostic Kit (sandwich assay) (Abbott Lab- oratories); Serono Diagnostic CEA Kit (double antibody assay) (Serono, Rome); and Phadebas CEA PRIST.

The antibody activity of the MAbs against CEA and NCA-2 was also measured by an enzyme-linked immunoadsorbent as-

SOLID PHASE MAB 27 CEA

FIG. 1. Principle of two-site MEIA. C and D denote groups of in- dependent epitopes on CEA. 3-GAL, ,3-galactosidase.

say (ELISA) (12) using antigen-coated microtiter plates (2 ,g/ ml) and a conjugate of rabbit anti-mouse Ig and alkaline phos- phatase.

RESULTS

MAbs Against CEA. None of the four MAbs crossreacted with NCA or BGP I (4). Their reactivity against NCA-2 was now investigated by using ELISA. Microtiter plates were coated with CEA or NCA-2, and the reactivities of different dilutions of the MAbs against the two antigens were compared. MAbs 9 and 27 reacted equally well with CEA and NCA-2, indicating that an- tigenic sites recognized by these two antibodies-i.e., epitopes A and C (4)-also are present in NCA-2. In contrast, the reac- tivity of MAbs 38S1 and 46 (both probably recognizing the same epitope, D) against the two antigens was markedly different. The titers against NCA-2 were 1/10 and 1/100 of those against CEA, respectively. Furthermore, MAb 38S1 gave a lower max- imal uptake against NCA-2 than against CEA. These findings indicate that the antigenic site in NCA-2 reactive with these antibodies is different from epitope D.

Two-Site MEIA. A sensitive assay was obtained when MAb 38S1 was used on the solid phase and MAb 27 was conjugated to the enzyme (Fig. 1). The standard curve is shown in Fig. 2. The assay was sensitive to as little as approximately 0.5 /g of CEA per liter and the measuring range was 0.5-250 /g/liter. Only at very high CEA concentrations (e.g., >5,000 /g/liter) was the "hook-effect" (13) observed.

2-

c ._=

0

1-

-5

4

3 c

0 2o o

- 2

-1

0.5 1.5 5 15 50 5-1 02 CEA, /g/liter

FIG. 2. Dose-response curve for CEA in the MEIA. The values ob- tained in the normal measuring range 0.5-200 ,g of CEA per liter are shown on the left. The curve on the right shows the so-called "hook-ef- fect" obtained at high CEA concentrations. Vertical bars give the stan- dard deviations calculated from four separate experiments run in du- plicate.

Medical Sciences: Hedin et al.

5.103 5-104

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3472 Medical Sciences: Hedin et al.

2.0 CEA /

c

o I Prostate o II / / / Ca. HCI04

1.0

Normal spleen 1CI04 lung HCIO4

| -- '

_ , ,,,(NCA GPI liver HCI04 NCA-2 ; Ib I iI I

1 10 10 2 103 104 105 106

Antigen, ng/ml

FIG. 3. Dose-response curves for CEA and related substances as well as for 1 M perchloric acid (HC104) extracts of normal and tumor tissues in the MEIA. Ca., carcinoma.

To determine the within- and inter-assay coefficients of vari- ation, 21 sera containing different concentrations of CEA were analyzed in quintuplicate on five different occasions. The sera were diluted 1:5 before assay. At low CEA levels (4 utg/liter) the interassay variation was 24.2%; at intermediate levels (24 ,ug/liter) the interassay variation was 12.9%; and at high levels (149 ,ug/liter) it was 2.8%. The within-assay variation was only slightly lower than the interassay variation.

The effect of dilution was investigated by assaying 10 serum

samples containing different concentrations of CEA in a 2-fold dilution series. The calculated CEA values were found to be independent of dilution in all sera tested.

The specificity of the MEIA was analyzed by investigating whether purified CEA-crossreactive substances and various tis- sue extracts gave uptake curves similar to those of CEA. The results are shown in Fig. 3 and Table 1. They can be sum- marized as follows: (i) All four individual CEA preparations in- vestigated gave identical uptake curves. (ii) Three individual

Table 1. CEA activity of purified CEA-related substances and extracts of normal and tumor tissues as determined by MEIA

CEA, g/g/mg dry weight Material

CEA, ,g/mg dry weight

CEA Preparation 47 Preparation 48 Preparation 50 Preparation 52

CEA,ow Preparation 27 Preparation 41 Preparation 45

NCA Preparation 41 Preparation 50

BGP I

NCA-2 Semipurified Purified Burtin

Orosomucoid

1,000 1,000 1,000* 1,000

850 615 800

<0.2 <0.2

<0.02

<0.02 <0.02 <0.02

<0.02

Liver metastases of colorectal carcinoma

Preparation 45t Preparation 48t Preparation 50t

Urinary bladder carcinomat

Prostate carcinomat

Normal Colont Spleent Lungt Livert Musclet Bilet Meconiumt Fecest Spleen* Livert Musclet

200 120 80

0.2

0.05

2 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002

* All values are calculated relative to this preparation (= 1,000 ,tg/mg). t Lyophilized 1 M perchloric acid extract. tLyophilized 0.9% NaCl extract.

Material

Proc. Natl. Acad. Sci. USA 80 (1983)

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Medical Sciences: Hedin et al.

Table 2. CEA activity of three CEA-related substances, NCA, BGP I, and NCA-2, as determined by MEIA and different commercially available CEA assays

CEA, ag/mg dry weight Assay CEA NCA BGP I NCA-2

MEIA 1,000* <1 <1 <0.5 Phadebas CEA PRIST 1,000 2 <1 250 Roche CEA RIA (Z-gel assay) 1,000 2 <1 200 Roche CEA EIA 1,000 <1 <1 1,000 Abbott CEA RIA 1,000 0.5 0.5 1,000 Serono CEA RIA 1,000 10 1 330 Sheep anti-CEA + MAb 27 1,000 50 1 330 Sheep anti-CEA + MAb 38S1 1,000 5 <1 10 * All values calculated relative to CEA (= 1,000 ,g/mg).

CEAlow preparations gave identical uptake curves closely sim- ilar to those of CEA. (iii) Purified NCA, BGP I, and, most im- portantly, NCA-2 did not react in the assay even at 10-50 x 103 times higher concentrations than those giving a significant uptake with CEA. (iv) With the exception of colon extract, per- chloric acid or saline extracts of normal tissues were nonreac- tive even when tested at very high concentrations. (v) Perchlo- ric acid extracts of colorectal, bladder, and prostate carcinoma contained CEA-active material in various concentrations.

The influence of CEA-crossreactive normal tissue compo- nents in four commercially available CEA immunoassays, which use conventional antibodies, and one assay that utilizes a com- bination of monoclonal and polyclonal antibodies were studied. As can be seen from Table 2, NCA-2 gave almost the same value as CEA (20-100%) in the five assays. The highest degree of CEA specificity was obtained in the assay utilizing two MAbs (e.g., MEIA). Interestingly, less specific assays were obtained when either of these antibodies (38S1 and 27) was used in com- bination with conventional (spleen-absorbed) anti-CEA anti- bodies.

Determination of CEA Values in Patients' Sera. Sera from 180 healthy individuals and patients with cancer and other dis- eases were studied. CEA values were determined with MEIA and in parallel with a conventional RIA (Phadebas CEA PRIST).

250-

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Proc. Natl. Acad. Sci. USA 80 (1983) 3473

Each serum was analyzed in duplicate on two separate occa- sions. Figs. 4 and 5 summarize the results (mean values are given). Healthy individuals (n = 40) gave CEA values below 5 .tg/liter in both assays. With 3 ,g/liter as the cutoff, 5 of 40 sera were positive in RIA as compared to 0 of 40 in MEIA. When sera from patients with nonmalignant liver disease were com- pared in the two assays a marked difference was seen. Thus, 26 of 45 sera (58%) had a CEA value above 5 Ag/liter in RIA as compared to 2 of 45 sera (4%) in MEIA. The difference in mean values was highly significant (mean values 6.0 and <3.2 Ag/li- ter in RIA and MEIA, respectively; P < 0.001). The lack of cor- relation between RIA and MEIA CEA values in these patients is also seen in the correlation diagram (Fig. 5).

Similarly, sera from patients with ulcerative colitis gave higher values in RIA than in MEIA. Five of 25 sera showed CEA val- ues >5 ag/liter in RIA as compared to 0 of 25 in MEIA. At the lower cutoff (3 ,ug/liter), 20 of 25 sera compared to 7 of 25 sera, respectively, had increased CEA. In contrast, when 60 sera from patients with carcinomas of colon, pancreas, lung, and breast were analyzed by the two assays no difference in CEA values was seen (correlation coefficient r = 0.95; Fig. 5). Sera from 10 patients with sarcoma, melanoma, and lymphoma were also analyzed by the two assays. The CEA values were generally higher in RIA than in MEIA.

DISCUSSION CEA is a large and complex macromolecule containing many different regions against which antibodies may be formed. With our series of eight MAbs (4), at least five different epitopes (A- E) in the peptide moiety of CEA were recognized. These ep- itopes were found in all CEA preparations from colorectal car- cinoma so far studied. None of these MAbs were absolutely specific for CEA. Similar or identical structures were present on one or several of the CEA-related substances, NCA-2, NCA, and BGP I. However, two antibodies, MAbs 38S1 and 46, rec- ognizing epitope D on CEA (4) reacted only weakly with NCA- 2 and not at all with NCA or BGP I. The structure responsible for the binding of MAbs 38S1 and 46 to NCA-2 can therefore not be identical to epitope D. On the other hand, epitopes A, B, C, and E are probably present also in NCA-2. Several other

*-

: ** 8* @0

I *

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O"

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5-

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00

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00000' 0

R I A M A 0 0R A M E R

RIA MEIA RIA MEIA RIA

.. 00 0

00 - - 0 - - -0 _

ee: eeeeee e ee eeeee

MEIA RIA MEIA RIA MEIA

Healthy Liver disease U.colitis Other

Carcinoma malignancies

FIG. 4. Distribution of CEA values in sera from healthy individuals and patients with malignant and nonmalignant diseases as determined by MEIA and RIA. The numbers of individuals (= serum samples) were as follows: healthy individuals, 40; nonmalignant liver disease, 45; ulcerative colitis, 25; carcinoma of colon, pancreas, lung, and breast, 60; and sarcoma, melanoma, and lymphoma, 10.

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3474 Medical Sciences: Hedin et al.

250- 0? individuals and patients with malignant and nonmalignant dis- 0 D eases by using the MEIA and in parallel a conventional RIA

]0 demonstrated that the MEIA was more specific for carcinomas

100- ? 1jg than was the RIA. This was essentially due to the MEIA giv- . [9000 ing lower CEA values than the RIA in sera from patients with

50- 00 nonmalignant disease. Carcinoma patients, on the other hand, c,,)~o a gave almost identical values in the two assays. It is now nec-

0^ ? essary to confirm these results on a larger sample of clinical w ? ywy material. Increased specificity for colorectal cancer was re-

a /0 cently reported with a mixed monoclonal/polyclonal assay (22). ,< lo- On the other hand, two other groups (23, 24) did not obtain w 1 - increased clinical specificity or sensitivity with assays using MAbs.

O A

5 o We thank Ms. A. M. Stromberg and Mr. R. Ghosh for excellent tech- + BA*? nical assistance. This work was supported by a grant from the Swedish

+ Cancer Society.

1. NIH Consensus Statement (1981) Br. Med. J. 282, 373-375. 2. Burtin, P., Chavanel, G. & Hirsch-Marie, A. (1973)J. Immunol.

111, 1926-1928. 1' i i i u u 3. Matsuoka, Y., Kuroki, M., Koga, Y., Kuriyama, H., Mori, T. &

1 5 10 50 100 250 Kosaki, G. (1982) Cancer Res. 42, 2012-2017. R IA CEA, g/ liter 4. Hedin, A., Hammarstrom, S. & Larsson, A. (1982) Mol. Immunol.

19, 1641-1648. FIG. 5. Correlation plot of CEA concentrations determined by MEIA 5. von Kleist, S., Chavanel, G. & Burtin, P. (1972) Proc. Natl. Acad.

and RIA in sera from healthy individuals and patients with malignant Sci. USA 69, 2492-2494. and nonmalignant diseases. *, Healthy individuals; o, carcinoma pa- 6. Mach, J.-P. & Pusztaszeri, G. (1972) Immunochemistry 9, 1031- tients; v, patients with sarcoma, melanoma, and lymphoma; A, patients 1034. with nonmalignant liver disease; +, patients with ulcerative colitis. 7. Hammarstr6m, S., Engvall, E. & Sundblad, G. (1976) in Health The line shows the relationship between RIA and MEIA values for car- Control in Detection of Cancer-Skandia International Symposia, cinoma patients (correlation coefficient = 0.95). Three carcinoma sera eds. Bostr6m, H., Larsson, T. & Ljungstedt, N. (Almqvist & Wik- were excluded from the analysis because they gave values outside the sell, Stockholm, Sweden), pp. 24-39. measuring range in both assays. 8. Hedin, A., Hammarstrom, S., Svenberg, T. & Sundblad, G. (1978)

Scand. J. Immunol. 8, Suppl. 8, 423-428. 9. Hammarstr6m, S., Svenberg, T., Hedin, A. & Sundblad, G. (1978)

groups (14-18) have produced MAbs against CEA. Some of these Scand. J. Immunol. 7, Suppl. 6, 33-46. antibodies did not react with NCA (14, 16). However, their 10. Svenberg, T., Hammarstrom, S. & Hedin, A. (1979) Mol. Immu- reactivity against NCA-2 has so far not been reported. nol. 16, 245-252.

When MAbs 38S1 and 27 were used in combination in an 11. Carlsson, J., Drevin, H. & Axen, R. (1978) Biochem.J. 173, 723- Whenzyme MAbs 38Say and 27 were used in c ombination inean. 737.

enzyme immunoassay a highly specific CEA test was obtained. 12. Carlsson, H. E., Lindberg, A. & Hammarstrom, S. (1972) Infect. Notably not even NCA-2 reacted in this assay. Apparently the Immun. 6, 703-708. use of two MAbs was essential for improving the specificity, 13. Miles, L. E. (1976) in Protides of the Biological Fluids, ed. Pee- because the combination of polyclonal and monoclonal anti- ters, H. (Pergamon, Oxford), Vol. 24, pp. 695-704. bodies gave less specific assays (Table 2). This was also true when 14. Accola, R. S, Carrel, S. & Mach, J.-P. (1980) Proc. Natl. Acad. Sci. the MAb that differentiates between CEA and NCA-2 was used USA 77, 563-566. . .oMAbinatdio wiath p a d. No f f 15. Kupchik, H. Z., Zurawski, V. R., Hurrell, J. G. R., Zamcheck, N. in combination with polyclonal antibodies. None of five com- & Black, P. H. (1982) Cancer Res. 41, 3306-3310. mercially available CEA assays tested were able to distinguish 16. Rogers, G. T., Rawlins, G. A. & Bagshawe, K. D. (1981) Br. J. between CEA and NCA-2. Cancer 43, 1-4.

When the two-site MEIA was used, a variety of normal tis- 17. Mitchell, K. F. (1980) Cancer Immunol. Immunother. 10, 1-5. sue extracts appeared to lack CEA activity, with the notable 18. Lindgren, J., Bang, B., Hurme, M. & Makela, 0. (1982) Acta Pa-

thol. Microbiol. Immunol. Scand. Sect. C 90, 159-162. exception of the colon sample investigated. It was taken 5 cm tho Microbiol Immunol. Scand. Sect. C 90, 159-162. exe p

tion tm . I i .ent unla whte . ino- 19. Fritsche, R. & Mach, J.-P. (1977) Immunochemistry 14, 119-127. from a known tumor. It is presently unclear whether it is nor- 20. Egan, M. L., Pritchard, D. G., Todd, C. W. & Go, V. L. W. (1977) mal colon CEA (19, 20), normal fecal antigen (NFA) (21), or tu- Cancer Res. 37, 2638-2643. mor CEA that is responsible for the reaction. 21. Kuroki, M., Koga, Y. & Matsuoka, Y. (1981) Cancer Res. 41, 713-

Occasional human sera contain antibodies reacting with mouse 720. immunoglobulin that may give rise to false positive values in an 22. Staab, H. J., Glock, S. & Hornung, A. (1982) Tumor Diagnostik

assay utilizing two antibodies from the same species. This dif- Ther. 3, 183-194. flassay utwlazsng

two antby

odes from the asame species ll o a 23. Buchegger, F., Phan, M., Rivier, D., Carrel, S., Accolla, R. S.

ficulty was overcome by performing the assay in 1% normal & Mach, J.-P. (1982)J. Immunol. Methods 49, 129-139. mouse serum. 24. Rogers, G. T., Rawlins, G. A., Keep, P. A., Cooper, E. H. & Bag-

The comparative analysis of CEA levels in sera from healthy shawe, K. D. (1981) Br. J. Cancer 44, 371-380.

Proc. Natl. Acad. Sci. USA 80 (1983)

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