CA1330545C - Characteristics and use of the anti-cea antibody produced by the hybridoma having the atcc number hbl0029 - Google Patents

Abstract

ABSTRACT

The present invention relates to a mouse monoclonal anti-CEA antibody with its hybridoma having the ATCC number HB10029. The anti-CEA antibody of the present invention could be used for tumor imaging or immunotherapy. The instant antibody can be used in a sandwich quantitative ELISA for CEA. It can be used for the purification of CEA.
There is provided diagnostic kits containing reagents performance of the assays including the solid phase immunoadsorbent containing the anti-CEA antibody, a solution of a second anti-CEA antibody and optionally, CEA
standards.

Description

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TITL~ OF THE INVEN~ION

Characteristics and use of the anti-CEA antibody produced by the hybridoma having the ATCC number HB10029.
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BACXGROUND OF THE INVENTION ~ -Carcinoembryonic antigen (CEA) is a tumor-associated antigen usually present with tumors of the colon. Gold P.
et al. first described CEA as a component of colon cancer (J. EXP. Med., (1965) 121, 439-462). CEA has since been investigated in a variety of other types of cancer as described by Goldenberg D.M. et al.(Ann. Intern. Med.
(1981), 94, 407-409), and Go V.L.W. and Zamcheck N. (Cancer (1982), 50, 2618-2623).

Immunoscintigraphy The use of polyclonal antibodies brings an important number of false negative and some false positive results, caused by an accumulation of the antibodies in the --~-reticulo-endothelial system. Even monoclonal antibodies used so far present the same problem, at a lesser extent.

Immunotherapy With radiolabelled antibodies no tumor regression could be obtained so far on patient as it was impossible to deliver enough radioactivity to the tumor (Mach J.P. et al, Schweiz. med. Wachr. (1987), 117: 1076-1086). An anti-CEA
antibody having a high specificity to CEA is needed.

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The Shoemaker et al. European patent application (EP-A-0225 709, Centocor Inc., published June 16, 1987) disclosed two monoclonal antibodies used in a sandwich assay. This assay eventhough more sensitive than the polyclonal assay, does not disclose the use of such an anti-CEA antibody for tumor imaging, nor immunotherapy.
These antibodies do not seem to have the property to detect malignancy in tissue.
Because of these drawbacks, there is not anti-CEA
antibody which could be used for immunohistochemistry for pathology.
An anti-CEA antibody which could be used to diagnose the patient having a malignant tumor would be highly desirable. Moreover, an antibody which could distinguish between colonic adenocarcinoma and carcinoid tumor of the colon would be of the greatest interest.

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1 3 ~ ri Surprisingly and in accordance with the present invention, there is provided a substantially specific mouse monoclcnal anti-CEA (carcinoembryonic antigen) antibody which is not immunoreactive with NCA (non-specific cross-reacting antigen). The main application of the anti-CEA antibody of the present invention is in the design of immunohistochemical tests and at distinguishing cancer from healthy tissue within the same organ, in routine tissue section. Further more, the present anti-CEA antibody can distinguish between colonic adenocarcinoma and carcinoid tumor of the colon.
The hybridoma producing the anti-CEA antibody of the present invention, has been deposited at the American Type Culture Collection under the number HB10029.
There is also provided in accordance with the present invention, a method of detecting a tumor in a tissue sample which consists in incubating the anti-CEA antibody with the tissue sample for a sufficient time to allow an immunologic reaction to occur.
Further, there is provided a sandwich method of measuring CEA in a tissue sample, which mainly consists of the steps:
a) a solid surface having bound thereto the anti-CEA
monoclonal antibody of the present invention;
b) contacting the antibody-coated surface with the .. .. .

4 ~ i 3 -~ 5 samples to be tested for a sufficient time to allow an immunologic reaction to occur; and c) contacting the coated surface of step b) with a second polyclonal or monoclonal anti-CEA antibody;

wherein the second monoclonal antibody is either directly or indirectly detected, so that when in operation the amount of CEA bound to the first anti~EA antibody is determined.

A diagnostic kit containing reagents for performing a sandwich type immunometric assay for CEA which consists in:

I) a solid phase immunoadsorbent having bound thereto the anti-CEA antibody; and II) an other anti~EA antibody which recogruzes at least a different epitope on CEA and which is labeled or indirectly detected.

Such an anti-CEA antibody provides a way to distinguish immunohistochemically between cancer and residual normal tissue within the same organ. For instance, in several cases of gastric, lung and breast cancer the tumor cells were strongly positive, whereas normal residual epithelial cells were negative within the range of detection of the system.

Therefore, the anti-CEA of the present invention has significant potentials in the 'in vitro' diagnosis of various forms of malignancies, especially those arising in the colon, lung, breast and stomach.

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~ 3 ~c) ~ 3 Other advantages of the present invention will be readily illustrated by referring to the following description.

BRIE~ DE8CRIPTION OF THE DRAWINGB

Figure 1 shows a titration curve of anti-CEA antibody towards solid phase immobilized CEA at a concentration of 1 ug/ml.
Figure 2 shows a binding curve of anti-CEA antibody towards ascending concentration of CEA immobilized on solid phase;
and Figure 3 shows the anti-CEA antibody specificity evaluated on Western blots.

DBTAII.ED DE8CRIPTION OF THE: INVENTION

The anti-CEA antibody of the present invention specifically binds to cancer of: breast, colon, lung, pancreas and stomach.
Generation ~n~ cloning of ~B 10029 hybri~oma Preparation o~ CEA u~e~ for im~unization CEA has been isolated from hepatic metastases from colorectal cancers. Purification proceeds through: water homogenization, perchloric acid extraction, ion-exchange, affinity and gel filtration chromatographies, and hydroxylapatite high pressure liquid chromatography.
Preparation of heat-treated CEA: heat at 85~C for 30 minutes in a solution of 10 mg CEA/mg of 0.1 M phosphate buffer, pH 5Ø r~
To ensure the monospecificity of idiotype, mice will ,_,=~.., ~
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be immunized with heat-treated CEA. Here, the working hypothesis is that a tumor marker evolved from a normal tissue or became re-expresses as a result of mutagenesis triggered by: temperature, denaturation, radiation, bacterial or vital toxins, chemicals, etc. This would cause either changes in primary amino acid sequences or in spatial configuration of affected gene products. Thus, normal individuals would have CEA sensitive to all of these treatments while cancer patients would not. By producing monoclonal antibodies to chemically or physically modified CEA, we would be able to obtain reagents that would interact with modified and thus cancerous CEA, but not with those epitopes on CEA that are common to both normal and tumorous tissues.
1. Immunization From 06-06-86 to 26-01-87 immunization with CEA: 4 i.p. or i.v. injections to administer a total of 65 to 145 ug of CEA. 03-08-87 injected i.p. 50 ug heat-treated CEA
and CFA. On the 10-08-87, the titer was > 1:80000. ;~
22-09-87 50 ug heat-treated CEA, i.v. (boosting before fusion).

2. Fu~ion -Day 0. 1.48 x 108 splenocytes from the immunized mouse have been fused with 1.48 x 107 myeloma cells. Cell viability after fusion was 24%. Ten plates of 96 wells were seeded with 104 cells/well containing already 104 splenocytes from a normal Balb/c female mouse. Total volume/well was 200 ul. Endothelial Cell Growth Supplement~ (Collaborative Research Inc.) was added to the fused cell suspension at a final concentration of 10 ug/well.

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Cells were in selective HAT medium (IMDM - 15% FBS-HAT).
Seven days after fusion, clones were fed with HAT medium.
Growth: 14 days after fusion 19% of wells had growing clones.

3. Screening of clones:
Test supernatant in ELISA, against CEA, HCEA
(heat-treated CEA) and NCA (non-specific cross-reacting antigen). Three clones have been found positive towards CEA and HCEA and negative towards NCA: FcP1D2, FcP2G2, FcP5F6. Supernatant was tested on Western blot against CEA
and NCA. Specificity towards CEA, no binding to NCA.

4. Expansion of clones:
From 1 well of 200 ul to 5 wells of 200 ul. After screening in ELISA (enzyme-linked immunosorbent assay) expansion of one of the 5 wells to 10 wells of 200 ul.
After screening in ELISA:
- subclone from 1 positive well - pool rest of positive wells for expansion. Transfer to 2 ml of HT medium and follow cell growth. Split regularly with IMDM-10% FBS medium, then with DMEM - 10%
FBS (see below, maintenance of HB10029 hybridoma).
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From one well of 200 ul, subclone by limiting dilutions to 0.5, 1 and 2 cells/well. Subcloning of FcP1D2 (10% growth), FcP2G2 (3.6% growth), FcP5F6 (5.7% growth).
Medium used to subclone was IMDM-15% FBS-HT. ECGS was added at final concentration 25 ug/ml.

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Aliquot cell suspension over a feeder layer of 5 x 104 splenocytes/well from normal female Balb/c mouse.
Subclone FcP2G2 - C1~ named C2 FcP2G2 - C7 named C3 FcP2G2 - E7 named C4 Master clones FcPlD2, FcP2G2, FcP5F6 and subclones C2, C3, C4 were frozen. After thawing, master ~lone FcP2G2 died, C2, C3 and C4 were pooled to give hybridoma ATCC number HB10029.

Pu~ification of the anti-CEA antibody ~ -1- 50% ammonium sulfate precipitation of cell culture supernatant, on ice. Precipitate was then collected by centrifugation, dissolved in PBS pH 7.4 and freed of (NH4)4SO4 by dialysis in the :.
same buffer.

2- Affinity chromatography on protein A-sepharose CL~
4B~Z9 of tissue culture ammonium sulfate precipitated. Protein A
binds through an Fc fragment of immunoglobulin and has high affinity for human, rabbit and some mouse IgG subclasses, except for mouse IgGl. Unfortunately, most of mouse monoclonal antibodies is of IgG1 subclass and will not bind to protein A. However, recen'dy developed binding buffers and new solid support matrices enable some of the mouse monoclonal IgGl to bind to protein A effectively (Bio-Rad~9, Pharmacia). A column of 8 ml of protein A was used.
Up to 40 mg of protein, equilibrated in binding buffer (3 M NaC1, 1.45 M glycine, pH 9.2) could be applied on column. The bound IgG
2 5 was eluted with Bio-Rad protein A elution buffer, pH 3.1. A yield of - . . ~ . . , . i i .

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12.5% was obtained.

F(ab')2 fragment isolation from anti-CEA IgGl The anti-CEA IgG was dialyzed in 0.1 M citrate buffer and protein concentration adjusted to 1.5 mg/ml. Freshly prepared stock solution of pepsin in citrate buffer was added to IgG solution in a ratio pepsin: IgG, 1:50. The mixture was incubated for four hours at 37C and reaction was stopped by the additon of 15 rnl of 1 M Tris buffer, pH 10.3 to 5 ml mixture.

Traces of undigested IgG and Fc fragment were removed by passage through protein A column.

Characteristics of the anti-CEA antibody Immunoglobulin class: IgGlK

Isoelectric point: pH 6.5 - pH 6.8 r..~:~.
The isoelectric point (pI) of HB10029 antibody was l 5 determined by isoelectrofocusing (IEF) in polyacrylamide gel on Phast system~) (Pharmacia). The typical heterogenous banding pattern of monoclonal antibodies often seen in IEF experiments was also observed here. Three distinct, closely migrating bands between pH 6.5 and 6.8 were detected. The origin of this rnicroheterogeneity is generally unknown, but differential loss of amino groups, fine differences in glycosylation, and interaction of the antibodies with the carrier ampholy~es have all been forwarded as possible explanations.

Molecular weight according to ME reduced SDS-PAGE (165.0 kd):

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heavy chain 53.0 kd, light chain 29.5 kd Hybridoma ATCC number: HB10029 : .
Immunologic speaAcity is as follows:
The anti-CEA monoclonal antibody is CEA-HCEA
specific. In ELISA, it reacts with purified CEA and HCEA. On Western blots, it does not cross-react with a known NCA (non~
specific cross-reacting antigen). It binds to CEA producing human adenocarcinoma cell lines (e.g. ATCC no. CL188) but does not bind to CEA non-secreting human adenocarcinoma cells by indirect l 0 immunofluorescence reaction. It stains neoplastic tissue on cryostat sections of human tumors but does not stain any normal tissues by indirect immunofluorescence. The antibody does not cross-react with any antigens on human blood cells when analyzed by fluoresceine activated cell sorter. ~.
The anti-CEA antibody specifically binds to cancer of:
breast, colon, lung, pancreas and stomach.
Immunoassays using the anti-CEA antibody to measure CEA ``

A) Inununometric assay: In this type of assay, a known amount of CEA or CEA in sample bind on a fixed amount of anti-CEA
2 0 antibody. Free and bound fractions are separated, and the amount of CEA analyte in the sample is determined from measurement of the amount of CEA in bound fraction. The standard curve is the %
of CEA bound to anti-CEA Mab (solid phase).

Reagents and sample may be added sequentially or 2 5 simultaneously. The assay can be in liquid phase or in solid phase. `
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In a liquid all the reagents are in solution. The reaction is terminated by incubating with a precipitating agent. As a precipitating agent there may be used the following:
anti mouse Ig anti-serum, immunobeads, immunolatex, pansorbin A, matrix bound protein A and G, polyethylene glycol, and immuno magnetic particles.
In a solid phase, the anti-CEA antibody is chemically or physically attached to an insoluble solid support or matrix. As a solid support there may be used the following: tube, bead, microtiter plate, microparticle, dipstick, disc, etc.
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B) The present anti-CEA antibody could be used in immunohistochemistry for pathology.

Immunohistochemistry techniques are well ~nown in the art and is used to diagnose a malignant tumor in a patient.
This antibody can also be used to distinguish between colonic adenocarcinoma and rarcinoid tumor of the colon.

C) Other assays can be designed using the anti-CEA antibody of the present invention over and abov~ the assay mentioned previously.

Purification of CEA could be achieved by an affinity column which consists of the anti-CEA antibody bound on a solid phase. Affinity chromatography could be achieved using a protocol described by Barbour H. M. (J. Immunol.
Methods (1976) Il, 15-23) The present invention will be more readily understood ~.' ~

by referring to the following example which is given to illustrate the invention rather than to limit its scope.

Immunospecificity of the anti-CEA

The immunologic potency of the anti-CEA antibody towards its target antigen, CEA was quantitated by antibody titration.

Briefly, antigens were immobilized on polyvinyl chloride plates (Titertek~9, Flow Laboratories) at a concentration of 1 ug/ml in 0.1 M sodium carbonate/bicarbonate buffer, pH 9.6 l 0 overnight at4C.

Before use, the plates were washed and incubated for 1 hr at 37C with 1% low-ht powder milk in Tris-saline (TS).

Antibody dilutions in TS containing 1% milk were incubated at 37C for one hour. After washes, the plate were l 5 incubated with peroxidase-conjugated anti-mouse IgG (Heavy &
Light) (Jacksons Lab~9) diluted in T~milk for 1 hr at 37C, then washed, and the enzyme activity revealed with o-phenylenediamine (Sigma~9) at 1 mg/ml in 0.1 M sodium citrate-citric acid buffer, pH 5.0, containing 0.03% H2O2.

2 0 The colour was read after 30 min. in an automatic reader using interference filter of 450 nm (Figure 1). Maximum Wnding of `
the anti~EA antibody is obtained with 1 ug/ml and 50% binding is obtained with 60 ng/ml of the antibody. ~ -The potency of anti~EA antibody was also on occasion ~ . ,, : :

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~-- 3 3 quantitated by constructing antigen concentration curves for which microtitration plates were coated with ascending concentrations of antigen (HCEA or CEA) and subsequently incubated with the anti-CEA antibody at the concentration equivalent to 50% of its maximum binding to the corresponding antigen, e.g. 60 ng/ml for CEA. A typical antigen concentration curve is presented in Figure 2.

Saturation of binding with 60 ng/ml of anti-CEA
antibody is obtained with 10 ug/ml of CEA and 50% binding of 60 ng/ml of anti-CEA antibody is obtained with 1 ug of C~A on solid 1 0 phase.

Results from figure 1 and 2 show that the anti-CEA
antibody has a strong binding potency to CEA, which varies proportionally to both reagent concentration.

EXAMPI,E II
Immunodetection on Western blots SDS PAGE (Sodium dodecyl sulfat~Polyacrylamide gel electrophoresis) was performed on 8% acrylamide-bisacrylamide (30:0.8) running gel with 5% stacking gel in a Tris-glycine discontinuous buffer system of Laemmli (Laemmli, UK (1970), 2 0 Nature 277:680-685) in a Bi~Rad vertical slab gel apparatus. Under reduced conditions, the protein samples were prepared by boiling for 5 min. in 4% SDS (w/v), 10% 2-mercaptoethanol (v/v), 20%
glycerol (w/v), 0.002% bromophenol blue. Proteins in the gel were stained with 0.25% Coomassie brilliant blue R in methanol/acetic 2 5 acid/water (44:10:46 by volume) and destained in the same solvent.

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Bio-Rad~) high and low molecular weight standards were applied for gel calibration.

The electrophorectically resolved proteins on SDS PAGE
were transferred at 35V for 4 hr. in 25 mM Tris, 192 mM glycine, 20% (v/v) methanol, pH 8.3 to nitrocellulose sheets according to the ~ ~.
method of Towbin (Towbin, H. et al. Proc. Natl. Acad. Sci.
1979;76:4350-54). The remaining binding sites on nitrocellulose were quenched by overnight incubation at 4C in 3% milk in TS.
The blots were then incubated for 3 hr at room temperature, while l 0 shaking, with the anti-CEA antibody diluted in T~1% milk and then for 2 hr with peroxidase conjugated anti-mouse IgG. The enzymatic reaction was carried out by overlaying the strips with 0-03% H22 in diaminobenzidine (Sigma) at 1 mg/ml TS. Each of the steps was followed by extensive washing with T~Tween 20.
l S Proteins electro-transferred to nitrocellulose were visualized by staining with 0.1% amido black in 45% methanol. 7% acetic acid and destained in the same solvent. -Pure CEA was separated by SD~PAGE and transferred to nitrocellulose where the immunoreactivity of the anti-CEA
antibody in tissue culture medium was evaluated. The culture supernatant of the anti-CEA antibody reacted only with the CEA
band (180 kd) and not with the NCA band (60kd) (Figure 3).

These results show that the anti-CEA antibody binds specifically CEA.
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EXAMPLE III
Immunohisto¢hemical screening of the anti-CEA
mono¢lonal antibody Mat~rial and Nethods Two hundred and forty samples of fixed human tissues, originating from 135 patients, were studied. The tumor cases were obtained mostly from the surgical Pathology Service of the Royal Victoria Hospital. Normal human tissues were mostly obtained from autopsies of young adults who died in motor vehicle accidents. Full clinical information and final pathological diagnosis were provided.

All tissues were routinely fixed in formalin and embedded in paraffin according to standard techniques.
Five micron sections were cut and immunostained with the anti-CEA of the present invention as described by Jothy et al. (Laboratory Investigation (1986) 54:108-117) with minor modifications. Briefly, after blocking of the endogeneous peroxidase activity, rehydrated sections were incubated sequentially with a 1/200 dilution of the anti-CEA of the present invention, peroxidase labelled anti-mouse Ig (Dako~), and peroxidase labelled swine anti-rabbit Ig (Dako2). Each step was followed by washes in Tris-saline buffer. The reaction was visualized by using the dimethylaminebenzidine substrate. Negative control consisted in replacing the anti-CEA of the present invention by NS-1 supernatant (ATCC no. TI 18) myeloma cells supernatant.

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A strong cytoplasmic and apical membrane staining was observed with 26/26 (100%) cases of colonic adenocarcinoma.
All tumor cells were reactive. The immunostaining was equally sensitive in the primary tumors and in various metastatic sites, such as liver, lymph nodes, lung, and bladder wall. No significant background staining of the stromal or non-epithelial cells was present using the 1/200 antibody dilution.
Two cases of carcinoid tumor of the colon were tested and both of them were negative, in keeping with the different origins since in these cases the tumor cells are derived from endocrine elements. Two colonic polyps were tested and both of them were positive.
Immunostaining of sections of normal colonic mucosa showed a membrane and occasionally cytoplasmic staining of the luminal surface and the upper third of the crypts.
one hundred and twelve cases of non-colonic neoplasms were tested. These included the most common forms of human malignancies. It was observed that 5/18 (28%) cases of breast adenocarcinoma were positive with the anti-CEA
antibody. In the case of lung cancers, 28/36 (78%) cases were positive. When lung tumors were divided between adenocarcinoma and squamous cell carcinomas are positive respectively. All cases of gastric adenocarcinomas (8/8) were positive and 3/8 (37%) of cases of pancreas carcinoma were positive.

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~ '. '~ ,~ ' ': ' ~ ~3 ~ . '' In summary, this immunohistochemical screening shows that the anti-CEA monoclonal antibody binds strongly to tumor-associated CEA. Based on published results, its specificity is better than the observed with the polyclonal antibodies to CEA. In particular, there is no immunohistochemical evidence of cross-reactivity with NCA. A remarkable feature of the anti-CEA of the present invention is its ability to distinguish immunohistochemically between cancer and residual normal tissue within the same organ. For instance, in æveral cases of gastric, lung and breast cancer the tumor cells were strongly positive, whereas normal residual epithelial cells were negative within the range of detection of the system. Therefore, the anti-CEA of the present invention has significant potentials in the 'in vitro' diagnosis of various forms of malignancies, especially those arising in the colon, 1 5 lung, breast and stomach.

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Claims (6)

1. A monospecific mouse monoclonal antibody to carcino-embryonic antigen (CEA) or the antigen binding fragment thereof, which does not react with non-specific cross-reacting family of antigens and said antibody being produced by the hybridoma bearing the ATCC number HB10029 and having an isoelectric point of pH 6.8, a molecular weight of SDS-PAGE of 165.0 kilodaltons, and belonging to IgG1K immunoglobulin class.

2. A method of detecting directly or indirectly, by means of immunohistopathology, a CEA-bearing tumor in a tissue sample which comprises the steps of:

contacting said anti-CEA antibody of Claim 1 with said sample for a sufficient time to allow an immunologic reaction to occur; and detecting the presence or absence of said immunologic reaction by detection means, said presence of the reaction indicating presence of said tumor, said detection means providing a direct detection when said anti-CEA antibody is labelled and said detection means anti-CEA antibody is labelled and said detection means providing an indirect detection when a second labelled antibody directed against said anti-CEA antibody is used.

3. An affinity column for the purification of CEA
which comprises a solid phase to which is bound the anti-CEA antibody of Claim 1.

4. A sandwich method of measuring CEA in an analyte liquid sample, which comprises the steps of:
a) providing a first anti-CEA monoclonal antibody according to Claim 1 attached to and coating a solid surface;
b) contacting said antibody coated surface with the samples to be tested for CEA content for a sufficient time to allow an immunologic reaction to occur;

c) contacting the coated surface of step b) with a second polyclonal or monoclonal anti-CEA antibody which recognizes at least a different epitope on said CEA; and d) detecting, directly or indirectly, the presence of said second antibody and, based upon said detection, determining the amount of CEA
contained in said sample and which is bound to said first anti-CEA antibody.

5. A diagnostic kit containing reagents for performing a sandwich type immunometric assay for CEA comprising:

I) a solid phase immunoadsorbent having bound thereto the anti-CEA antibody of Claim 1;
II) a second anti-CEA antibody which recognizes at least a different epitope on CEA and which is detected by means of a label; and III) a CEA standard protein.

6. A method of making the antibody of Claim 1 which comprises the following successive steps:
a) isolating CEA from cancer tissues;
b) heating said isolated CEA at 85°C for 30 minutes in a buffer pH 5.0;

c) immunizing mouse with the said heat-treated CEA:

d) fusing splenocytes from said immunized mouse with myeloma cells, in order to get hybridoma showing positive binding toward CEA and negative binding to NCA (non-specific cross-reacting antigen);
f) expanding said hybridoma; and g) purifying said antibody secreted by said hybridoma in a culture medium by precipitation with ammonium sulfate, dialysis against a buffer pH 7.4 and chromatography on an affinity column binding through a fragment of said antibody common to IgG1K
immunoglobulin class.