CA1339469C - Production and characteristics of anti-anti-cea antibody - Google Patents

Abstract

The present invention relates to anti-anti-CEA antibody against a mouse monoclonal anti-CEA antibody with its hybridoma having the ATCC number HB10029. The anti-anti-CEA antibody of the present invention can be used for immunizing against cancer a host as well as for cancer therapy. There is provided assays for measuring CEA in samples using the anti-idiotypic antibody of the present invention. A method of purifying anti-CEA antibody is also provided. Diagnostic kits adapted to be used with the assays are also described. An hybridoma cell line of an anti-anti-CEA antibody of the present invention has been deposited at the ATCC under the accession number HB10469.

Description

1339~69 TI~LE OF THE INVENTION

Production and characteristics of an anti-anti-CEA
antibody.

BACKGROUND OF THE INVENTION

The past 15 years have seen extensive clinical investigation of cancer immunotherapy. Varied biologic response modifiers have been used, including specific immunization with tumor cells or fractions thereof and nonspecific immunization with such materials as bacterial vaccines, thymic extracts, WBC fractions, l 0 and several chemical immunoadjuvants. These attempts at immunotherapy often have involved patients with far-advanced cancer, having larger tumor-cell burdens and impaired immune mechanisms that had little chance of being effectively aug~nented.
Investigation of these approaches with patients who have relatively poor prognoses at a time when their disease is limited seems justifiable at present; eg, in a patient with metastatical renal carcinoma in the lung whose primary tumor has been surgically resected. Such immunologic approaches to cancer are experimental.
The possibility exists of shifting the delicate balance between factors stimulating and inhibiting immunologic defenses against a tumor in an unfavorable direction.

Jerne's Network Theory In 1974, Niels Jerne (Jerne N.K., Ann Immunol. tParis), 1974: 125c:373-89) proposed a theory which pictured the immune 2 5 system as a network of interacting idiotypes. It had been known ~ .

before that antigens are recognized by "variable" regions of antibody molecules called paratopes. Parts of these variable regions, however, can function as antigens per se and are known individually as "idiotopes" or collectively as idiotypes. The S antibodies reacting with a group of idiotopes are called "anti-idiotypic antibodies". Anti-idiotype antibodies will themselves carry idiotopes which, in turn, will be recognized by other anti-idiotype antibodies. Jerne's theory postulates that the production of anti-idiotype antibodies is a normal component of the immune response. Exposure to an antigen invokes not only an antibody response to that antigen but also a cascade of anti-antibody (anti-Id) responses. Jerne emphasized the dual character of antibody molecules and the fact that for every paratope a complementary fitting idiotope on another antibody molecule can be found, and vice versa. Some idiotopes may be stereochemically similar to the epitope on the antigen against which the antibody was originally directed and such idiotopes are designated as an internal image of the antigen. They would mimic the nominal antigen by fitting into the actual antigen-binding site of idiotype and thus tending to 2 0 expand the idiotype production.

Anti-Idiotypic Antibodies as Vaccines The concept of anti-idiotypic vaccine relates to the generation of immune response to an antigen (e.g. viruses, bacteria, parasites, tumor markers) by immunization of the host with an 2 5 immunoglobulin that defines a specific region on the anti-pathogen antibody molecule, and in turn, engenders an antibody that mimics, 3 13~$69 in its function, such an anti-pathogen antibody. In other words, the aim of immunization with anti-idiotypic antibody (Ab2) is to produce an antibody (Ab3) reacting with the antigen in such a way that it will protect the host from the disease. In principle, a S monoclonal Ab2 detecting an inters~e.ies cross-reactive idiotope associated with the immune response against, for example, a neutralization domain of a viral epitope, or possessing an "internal image" of such an epitope, would represent the ideal anti-idiotypic vaccine. The internal image anti-idiotype could thus substitute for the nominal antigen and induce protective immunity. In most cases, anti-idiotypes that engendered antigen reactive Ab3 were cross-reactive for idiotypes common to many antibody preparations generated in different animal species. Until now the applicability of Ab2 vaccines seemed restricted to internal image Ab2 or anti-cross reactive idiotope Ab2 but recently the expansion of the availability repertoire by activation of silent clones bearing private idiotopes has been reported.

What make anti-idiotypic vaccine so special is the fact that for a number of human and animal diseases antigens are still unknown or they cannot be prepared in sufficient amounts or in proper immunogenic form. Some of the common pathogens are known for antigenic variation and high toxicity, and polysaccharide or glycoprotein antigens cannot be substituted with synthetic peptides.

2 5 Utilization of anti-idiotype antibodies for tumorimmunotherapy is based on the concept of stimulation of the tumor 1~9469 host's own response against the tumor. Such an anti-idiotypic antibody can be used as surrogate tumor-associated antigens (TAA
like CEA) as an alternative to administration of CEA itself. Indeed, an active immunization of the patient with CEA would be efficient, but the immune system is usually tolerant to T.A.A. as it is recognized as a self component (Greene, M.I., Contemp. Top.
Immunobiol.(1980), 11:81 116; Mc Bride, W.H. et al., Br. J. Cancer (1986), 53:707-711). Moreover, large amounts of purified CEA, free from contamination with viruses and viral products would be l 0 necessary. The passive administration of monoclonal antibodies againt tumor-associated antigens has been used in the therapy of human cancer as described by Sears H.F. et al., a Biol. Response Modif.(1984), 3:138-150). But this approach is limited since the antibodies are usually of murine origine, leading to the production l 5 of human anti-mouse immunoglobulins that can hamper the efficacy of the treatment.

Active immunization of patients with an anti-idiotypic antibody is of a great interest. Moreover, the induction of protective immunity against metastasis on patients whose primary tumor has 2 0 been removed by surgery or on patients in highly risk group would also be of a great interest.

It would be highly desirable if there could be provided an anti-idiotype antibody which could be used for immunizing a host against canoer as well as for canoer therapy.

SUMMARY OF THE INVENTION

Surprisingly and in accordance with the present invention, there is provided an anti-anti-CEA antibody, or a fragment thereof, raised against a substantially specific mouse monoclonal antibody to CEA
(carcinoembryonic antigen) which is not immunoreactive with NCA (non-specific cross-reacting antigen).

An hybridoma cell line of an anti-anti-CEA
antibody of the present invention was deposited at the American Type Culture Collection under the accession number HB10469 on May 25, 1990.

The anti-idiotypic antibody of the present invention can be used for immunizing against cancer a host as well as for cancer therapy.

There is also provided a method for the purification of anti-CEA antibody which consists of the anti-idiotypic antibody solid phase bound and adapted to be used in affinity chromatography.

There is also, a competitive method of measuring CEA in patients which consists of incubating at least the anti-CEA antibody, the anti-idiotypic antibody, and an unknown amount of CEA analyte present in a patient's body fluid. The anti-CEA antibody or the anti-idiotypic antibody is either directly or indirectly detected. In operation, the amount of CEA analyte, in a patient's body fluid is determined by comparing the extent to which the anti-idiotypic antibody is bound to the anti-CEA antibody or vice-versa with a calibration curve obtained with a known amount of said anti-D

~339469 idiotypic antibody.

In one embodiment the competitive method is a solidphase assay wherein the anti-CEA antibody or the anti-idiotypic antibody is solid phase bound.

In another embodiment the competitive method is a liquid phase assay which requires incubating also with a precipitating agent.

A diagnostic kit including reagents for competitively measuring CEA in a sample such as:
I) a solid surface having bound thereto a first anti-CEA
antibody;
II) a known amount of labelled anti-idiotypic antibody;
and III) a known amount of the anti-idiotypic antibody, whereby a standard curve can be obtained.

There is also another diagnostic kit including reagents for measuring CEA in a sample such as:
I) a solid surface having bound thereto the anti-idiotypic antibody;
II) an anti-CEA antibody which is labelled or indirectly fletecte~l; and III) a known amount of anti-idiotypic antibody, whereby a standard curve can be obtained.

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

s-~ ~

1~9469 IN THE DRAWINGS

Figure lA is a binding curve of the purified anti-idiotypic antibody to coated anti-CEA F(ab')2 antibodies (2ug/ml) or polyclonal rabbit anti-CEA IgG (3ug/ml);

Figure lB is the control curve using normal mouse F(ab')2 (2ug/ml) or normal rabbit IgG (3ug/ml);

Figure 2A is the inhibition of anti-CEA F(ab')2 binding to CEA by anti-idiotypic antibody IgG (FLP12E2) of 3 different concentrations;

Figure 2B is the control curve using maximal concentration of an unrelated monodonal antibody or normal mouse IgG;

Figure 3 is the inhibition effect of purified anti-idiotype IgG on CEA
binding to anti-CEA F(ab')2 (2ug/ml) on solid phase; and Figure 4 is the inhibition effect of 25 ug/ml of CEA or NCA on anti-idiotypic antibody binding to anti-CEA F(ab')2 on solid phase l 5 (2ug/ml).

DETAILED DESCRIPTION OF THE INVENTION

Preparation The anti-idiotypic antibody of the present invention is generally obtained by the following steps:

2 0 a) immunizing a host with the anti-CEA antibody of the hybridoma having the ATCC number HB 10029;

8 ~3~9469 b) collecting the host lymphocytes isolated from the spleen of the politeal lymph nodes;

c) when collecting the lymphocytes, fusing with myeloma cells to get a hybridoma cell line producing anti-anti-CEA
S monoclonal antibodies.
Results 1) The results from direct binding experiments using the anti-anti-CEA monoclonal antibody show that the anti-anti-CEA
monoclonal antibody (Mab) is specific to the anti-CEA Mab used for 1 0 immunization;

2) The results from inhibition assays using the anti-anti-CEA show that this anti-idiotype antibody and CEA share related epitopes.

The anti-anti-CEA monoclonal antibody binds specifically anti-CEA F(ab')2 framework where CEA binding site is involved.

As a composition for stimulating the immune system of a host, the anti-anti-CEA can be used in association with a pharmaceutical acceptable carrier. The expression "pharmaceutical 2 0 acceptable carrier" means a carrier for example to be administered intraveneously, intradermally or subcutaneously for example an injectable aqueous solution, sterile and isotonic, a saline solution (0.9%), alum and the like.
The expression "body fluid" means any body secretion that may contain CEA for example blood, plasma, urine, lymph, cerebrospinal fluid and the like.

Characteristics of the anti-CEA antibody Immunoglobulin class: IgG1 K
Isoelectric point: pH 6.5 - pH 6.8 Molecular weight according to ME reduced SDS-PAGE (165.0 kd):
heavy chain 53.0 kd, light chain 29.5 kd Hybridoma ATCC nwnber: HB10029 Immunologic specificity is as follows:
l 0 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. CL 188) but does not bind to CEA non-secreting human adenocarcinoma cells by indirect 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 2 0 fluoresceine activated cell sorter.
The anti~EA antibody specifically binds to cancer of:
breast, colon, lung, pancreas and stomach.
Immunoassays using monoclonal anti-idiotypic antibody to measure CEA, as a replacement of CEA

A) Competitive assay: In this type of assay, known amounts of unlabelled anti-idiotypic antibody or CEA in sample compete with ~339469 known amount of labelled anti-anti-CEA antibody for binding sites on a limiting amount of anti-CEA antibody. Free and bound fractions are separated, and the amount of CEA analyte in the sample is determined from measurement of the amount of anti-S idiotypic antibody in bound fraction. The amount of bound anti-idiotypic antibody is inversely proportional to the amount of analyte in the sample. The standard curve is the % of labelled anti-idiotype Mab bound to anti~EA (solid phase) vs concentration of unlabelled anti-idiotype (in solution). At a 100% binding, there is no unlabelled anti-idiotype in solution.

The assay can be in liquid phase or in solid phase. In a liquid phase, 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 2 0 solid support there may be used the following: tube, bead, mi.. oliler plate, micropartide, dipstick, disc, etc.

B) Inhibition assay: In this assay, the inhibition of binding of anti-CEA antibody to anti-idiotypic antibody by CEA in sample is measured. The standard curve is the % of anti-CEA Mab bound to anti-idiotype (solid phase) vs concentration of anti-idiotype (in solution). At a 100% binding, there is no anti-idiotype in solution 1339~69 (or no CEA in sample). The anti{~EA Mab is directly or indirectly detected.

The expression "directly detected" is referred to as any label to be used according to the type of immunoassay chosen.

S The expression "indirectly detected" is referred to as another antibody (which recognizes the anti-idiotypic antibody) labelled adapted to be used according to the type of immunoassay chosen.

C) Other assays can be designed using the anti-idiotypic antibody of l 0 the present invention over and above the assay mentioned previously.

There is also provided, a method for the purification of anti-CEA antibody which consists of the anti-idiotypic antibody solid phase bound and adapted to be used in affinity chromatography.
Affinity chromatography is as described by Barbour H. M. (J.
Immunol. Methods (1976), ~,1~23) Vaccine or anti-cancer ~eatment It is well known that if an antibody recognizes a certain molecule, for example CEA, its anti-idiotypic antibody would be useful in stimulating the immune system of a host against this certain molecule.

According to previous studies on patients, the anti-anti-CEA antibody of the present invention could be used as an anti-cancer vaccine.

In the Koprowski's experiment, the inoculation of patients with mouse monoclonal antibody which binds to gastrointestinal cancer cells resulted in the production of an anti-idiotypic antibody. Such an anti-idiotypic antibody binds specifically to the binding site of monoclonal antibody to the gastrointestinal cancer cells. The inoculated patients, which developed the anti-idiotypic antibody, improved clinically and even had long remission from their disease (Koprowski H. et al, Proc. Natl. Acad. Sci. USA, (1984), ~, 216-219).

In another study, thirty patients suffering from advanced colorectal carcinoma were treated with polyclonal goat anti-idiotypic antibodies. Among those, six patients showed partial clinical remission and seven patients showed arrest of metastases following immmunotherapy (Herlyn D. et al, Proc. Natl. Acad. Sci. USA (1987), 1 5 ~, 8055-8059).

In the recent study of Mellstedt et al (Med. Oncol. &
Tumor Pharmacother. (1989), vol. ~, no.l 99-107), a mouse monoclonal antibody was used for the treatment of patients suffering from metastatic colorectal carcinoma. Patients developed anti-idiotypic antibodies (Ab2) and some patients developed anti-anti-idiotypic antibodies (Ab3). The patients having the best clinical response developed the highest titers.

Taken together, these results provide good evidence for efficacity of anti-idiotypic antibodies in treatment and vaccination 2 5 against corresponding cancer.

1339~69 The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

Example I

S Generation of anti-idiotypes monoclonal antibodies - Antigen:
Anti CEA IgG, ATCC deposition number: HB10029 on February 8,1989.
Anti CEA IgG was conjugated to heat-inactivated tuberculin PPD
(protein purified derivative), using gluteraldehyde. Products and method for conjugation from Cambridge Research Biochemicals Ltd kit (catalogue No: CK-07.40 000) 100 ul of conjugate is equivalent to: 26.7 ug anti CEA IgG and 50 ug tuberculin-PPD.

- Immunization:
Day-14: 10 female Balb/c mice were injected with 100 ul conjugate l 5 emulsified with an equal volume of complete Freund's adjuvant into two foot pads, 100 ul per site.
Day-3: injection of 100 ul conjugate into both foot pads (50 ul/site).

- Fusion (Day 0):
a) - collect blood from orbital vein from each mouse.
2 0 b) - collect popliteal lymph nodes from 10 mice and pool.
c) - fuse and plate 2.2x104 live cells/well into 96 wells plates (total of 12 plates).

- Post-fusion Day+7: each well fed with 100 ul of HAT medium.

14 13~469 Day+11: 95% growth.
Day+12: ELISA on supernatants against anti-CEA F(ab')2 (HB10029) and normal mouse F(ab')2: all dones were positive for HB10029 idiotype only, 144 of them are strongly positive.
Clones fed with 100 ul of HAT medium.
Day+14: feeding of clones with 100 ul of HT medium.
135 strongly positive dones expanded.
Day+17: non-expanded clones directly frozen in fusion plates.
Day+21: ELISA on supernatants from expanded clones against normal l 0 and anti~EA rabbit IgG (134 out of 135 clones grew):
- 7 clones were positive for both antigens (eliminated) - 127 clones were negative for both antigens.
Day+22: ELISA on supernatants from 127 expanded clones, against HB10029 F(ab')2 (idiotype): 124 dones were still positive.
1 5 Day+23: cells from 124 clones were frozen. Cells from 20 of these clones were expanded.
Day+27: competition ELISA on supernatants from 20 clones: idiotype on solid phase, competition CEA+anti-idiotype, mesure CEA %
inhibition varied from 4 to 98%. Selection of 7 clones for best 2 0 competition: FL P3 A1, FL P4 AB, FL P4 G6, FL P5 A9, FL P5 G12, FL P6 A4,FLP12E12.
Day+28: expansion of 20 clones.
Day+42: direct ELISA of 17 supernatants against idiotype: HB10029 F(abl)2-2 5 positive clones = FL P1 A6, FL P1 H5, FL P2 A9, FLP2H12,FLP3A1,FLP4AB,FLP4G6.
FL P5 A9, FL P6 A4, FL P6 H5, FL P8 A3, FLP12E2.

4 clones were negative, 1 clone was weakly positive.
Day+56: freezing of cells from FL P1 H5, FL P2 A9, FL P2 H12, FL P3 H7, FL P4 A8, FL P4 G6, FL P6 G5.
Day+57: freezing of cells FL Pt A9, FL Py A4, FL P12 E2 and from negative clone FL P9 G1 (control).
Continue to grow cells from FL P5 A9, FLP6A4,FLP12E2.
Day+64: start IgG isolation from FL P12 E2 cell culture supernatant.

1 0 Example II

IgG purification of anti-anti-CEA monoclonal antibody.
1) Tissue culture supernatant of FL P12 E2 hybridoma was five fold concentrated by ultrafiltration on PM 30 membrane and precipitated with 50% saturated ammonium sulfate, pH 6.8 for 60 1 5 minutes at 4~C, stirring. The precipitate was collected by centrifugation at 8000 rpm for 20 minutes and resuspended in PBS. It was dialyzed against PBS (phosphate buffer saline) for two days with 4 buffer changes.
2) IgG was specifically purified from above solution by 2 0 affinity chromatography: up to 50 mg of protein were applied on a 10 ml column of anti-CEA HB10029 monoclonal antibody coupled to Sepharose 4B, equilibrated in PBS. After a 30 minutes incubation period at room temperature, the unbound material was eluted with 50 ml of PBS. Anti-anti-CEA IgG was then eluted with 25 ml of 3M
2 5 NaSCN, pH 7.4. Eluted fractions were pooled, concentrated on PM 30, and dialysed against PBS. IgG concentrations were determined by 1339~69 lowry assay Example III

Immunos~e~ilici~y of the anti-anti-CEA antibody The immunoreactivity of the anti-anti-CEA monoclonal antibody (anti-anti-CEA Mab ATCC number HB10469) towards is target antigen (anti-CEA Mab ATCC number HB10029) and other anti-CEA
antibodies was quantitAte~l by antibody titration in ELISA.

Idiotype ELISA
Idiotype or other antibodies to be tested were immobilized on polyvinyl chloride plates (Titertek~, Flow Laboratories) at a concentration of 2 ug/ml in 0.1 M sodium carbonate/bicarbonate buffer, pH 9.6 overnight at 4~C. Before use, the plates were washed and incubated for 1 hr at 37~C with 1% low-fat powder milk in Tris-saline (TS). Antibody dilutions in TS containing 1% milk were incubated at 37~C for 1 hr.

After washes, the plates were incubated with peroxidase-conj1lgatetl anti-mouse IgG, Fc or anti-rabbit IgG F(ab')2 (Jacksons Lab) diluted in TS-milk for 1 hr at 37~C, then washed, and the enzyme activity revealed with o-phenylenediamine (Sigma~) at 1 mg/ml in 0.1 M sodium citrate-citric buffer, pH 5.0, containing 0.03% H202. The colour was read after 30 min. in an automatic reader using in~elfel~l.ce filter of 450 nm.

The results are shown in Figure 1 and demonstrate that the anti-idiotypic antibody is specific to the immunizing anti-CEA
antibody.

i ,~, ~ ... .

17 13~Y469 Exarnple IV

INHIBITION ASSAYS
These assays were conducted to determine whether or not the anti-idiotype binds to a determinant located within the CEA-anti-CEA binding site.
A) Inhibition of CEA binding to anti-CEA Mab by anti-idiotype IgG

The anti-CEA F(ab')2 (2 ug/ml) was immobilized on the ELISA plate and non-specific binding sites were blocked with 1% TS-milk as in the procedure of Example III. CEA and anti-idiotype l 0 supernatant or anti-idiotype purified IgG were incubated together for 1 hour at 37~C. After washing, plates were subsequently incubated with 5 ug/ml of rabbit anti-CEA IgG and peroxidase-conjugated goat anti-rabbit IgG F(ab')2. Assay was terminated as in the procedure of Example m.

l 5 The results are shown in figure 3 and demonstrate that the anti-idiotypic antibody is a potent inhibitor of the binding CEA to anti-CEA antibody. Therefore, the anti-idiotypic antibody shares epitopes with CEA.
B) Inhibition of the anti-CEA Mab binding to CEA by anti-idiotype IgG

2 0 CEA was immobilized on ELISA plates at a concentration of 0.5 ug/ml. Anti-CEA F(ab')2 and anti-idiotype IgG were incubated simultaneously for one hour at 37~C and bound anti-CEA F(ab')2 was measured using a peroxidase-conjugated goat anti-mouse F(ab')2.
Other steps of ELISA were conducted as in Example m.

133g469 The results are shown in figure 2 and demonstrate that the anti-idiotypic antibody is a potent inhibitor of binding of the anti-CEA
antibody to CEA. Therefore, the anti-idiotypic antibody shares epilo~es with CEA.
S C) Inhibition of anti-idiotype IgG binding to the anti-CEA F(ab')2 by CEA

Anti-CEA monoclonal antibody was immobilized on ELISA
plates (2 ug/ml of F(ab')2). Anti-idiotype and CEA were incubated simultaneously for one hour at 37~C. Bound anti-idiotype was detected by peroxidase-conjugated goat anti-mouse IgG, Fc fragment. Other steps of ELISA were conducted as in Example m.

The results are shown in figure 4 and demonstrate that the CEA but not NCA (60 kd) is a potent inhibitor of the binding of the anti-idiotypic antibody to the anti-CEA antibody. Therefore, the anti-1 5 idiotypic antibody shares epitopes with CEA.

Claims (8)

1. An anti-idiotypic antibody, or fragment thereof, raised against a mouse monoclonal anti-carcinoembryonic antigen (anti-CEA) antibody, which is not immunoreactive with any non-specific cross-reacting antigens and said anti-CEA antibody being produced by the hybridoma having the ATCC number HB10029.

2. The anti-idiotypic antibody of claim 1, wherein said antibody is a monoclonal antibody produced by the hybridoma having the ATCC number HB10469.

3. A composition for stimulating the immune system of a host against cancer, which comprises a pharmaceutical amount of the anti-idiotypic antibody of claim 1 in association with a pharmaceutically acceptable carrier.

4. A method for the purification of anti-carcinoembryonic antigen (anti-CEA) antibody which comprises: 1) contacting a material containing the anti-CEA antibody to be purified, with the anti-idiotypic antibody of claim 1 for a time sufficient to obtain a complex anti-idiotypic antibody:anti-CEA antibody, said anti-idiotypic antibody being bound to a solid phase and adapted to be used in affinity chromatography; 2) eluting material not complexed with said anti-idiotypic antibody;
and 3) eluting the anti-CEA antibody from the complex, thereby purifying the anti-CEA antibody.

5. A competitive method of measuring carcinoembryonic antigen (CEA) in patients which comprises incubating at least an anti-CEA antibody, the anti-idiotypic antibody of claim 1, and an unknown amount of CEA analyte present in a patient's body fluid;
wherein at least one member selected from the group consisting of anti-CEA antibody and anti-idiotypic antibody is either directly or indirectly detected, whereby the amount of CEA analyte, in a patient's body fluid is determined by comparing the extent to which the anti-idiotypic antibody is bound to the anti-CEA antibody with a calibration curve obtained with the percentage of anti-CEA bound to anti-idiotypic against a concentration of anti-idiotypic present.

6. The competitive method according to claim 5, wherein one member selected from the group consisting of anti-CEA antibody and the anti-idiotypic antibody is bound to a solid phase.

7. A diagnostic kit for measuring carcinoembryonic antigen (CEA) in a sample adapted to be used according to the method according to claim 5 comprising:
I) a solid surface with an anti-CEA antibody bound thereto;
II) a known amount of the anti-idiotypic antibody of claim 1; and III) a known amount of the anti-idiotypic antibody of claim 1, which is labelled.

8. A diagnostic kit for measuring carcinoembryonic antigen (CEA) in a sample adapted to be used according to the method according to claim 5 comprising:
I) a solid surface having bound thereto an anti-idiotypic antibody of claim 1;
II) an anti-CEA antibody which is labelled or unlabelled; and III) a known amount of the anti-idiotypic antibody of claim 1 for use in obtaining a standard curve.