NOVEL ASSAY

Abstract

A method is described for the detection of at least two of cytokeratins 8, 18 and 19 in a sample. It is practiced by contacting the sample with a solid phase having a first antibody with specificity for cytokeratin 8, a second antibody with specificity for cytokeratin 18 and, optionally, a third antibody with specificity for a first epitope of cytokeratin 19 bound to it and allowing cytokeratins in the sample to bind to the bound antibodies to form complexes. The complexes are then contacted with a first labelled antibody with specificity for a dimer of cytokeratin 8 and 18 and optionally a second labelled antibody with specificity for a second epitope of cytokeratin 19 and allowing the labelled antibodies to bind to the complexes. The labelled antibodies bound to the complexes are then detected. A method for quantitative determination of soluble fragments of at least two of cytokeratin 8, 18 and 19 in a sample and a kit are also described.

Claims

1. A method for detecting soluble fragments of at least two cytokeratins selected from the group consisting of cytokeratin 8, 18 and 19 in a fluid sample of a human subject, comprising the steps of contacting said fluid sample with a solid phase having immobilized thereon a first antibody having specificity for cytokeratin 8, a second antibody having specificity for cytokeratin 18 and optionally a third antibody having specificity for a first epitope of cytokeratin 19; allowing soluble fragments of cytokeratins to bind to said first, second and optionally third antibodies thereby forming complexes; contacting said complexes with a first labelled antibody having specificity for a dimer of cytokeratin 8 and 18 and optionally a second labelled antibody having specificity for a second epitope of cytokeratin 19; allowing said labelled antibodies to bind to said complexes; and detecting said labelled antibodies bound to said complexes.

2. The method according to claim 1, wherein said antibodies are monoclonal antibodies.

3. The method according to claim 1, wherein said first labelled antibody has specificity for the -helix 2B 2 (aa 414-429 as set forth in SEQ ID NO:1) of cytokeratin 18, and, if present, said second labelled antibody has specificity for the -helix 2B 2 (aa 311-335 as set forth in SEQ ID NO:2) of cytokeratin 19.

4. (canceled)

5. The method according to claim 3, wherein said first labelled antibody is M21, and, if present, said second labelled antibody is A53-B/A2.

6. (canceled)

7. The method according to claim 1, wherein said first immobilized antibody has specificity for the -helix 2B (aa 340-365 as set forth in SEQ ID NO:3), said second immobilized antibody has specificity for the -helix 2B (aa 320-350 as set forth in SEQ ID NO:4), and, if present, said third immobilized antibody has specificity for the -helix 2B (aa 340-370 as set forth in SEQ ID NO:5).

8. The method according to claim 7, wherein said first immobilized antibody is 6D7, said second immobilized antibody is 3F3, and, if present, said third immobilized antibody is IDLC4.

9. The method according to claim 1, wherein said first antibody having specificity for cytokeratin 8 constitutes 20-40% of total immobilized antibody, said second antibody having specificity for cytokeratin 18, constitutes 5-15% of total immobilized antibody, and said third antibody having specificity for cytokeratin 19 constitutes 50-70% of total immobilized antibody.

10. The method according to claim 9, wherein said first antibody having specificity for cytokeratin 8 constitutes 30% of total immobilized antibody, said second antibody having specificity for cytokeratin 18, constitutes 10% of total immobilized antibody, and said third antibody having specificity for cytokeratin 19 constitutes 60% of total immobilized antibody.

11. The method according to claim 1, wherein the soluble fragments of cytokeratin 8 and 18 in the fluid sample are detected by a method comprising the steps of: contacting said fluid sample with a solid phase having immobilized thereon a first antibody having specificity for cytokeratin 8, and a second antibody having specificity for cytokeratin 18; allowing soluble fragments of cytokeratins in said sample to bind to said first and second antibodies thereby forming complexes; contacting said complexes with a first labelled antibody having specificity for a dimer of cytokeratin 8 and 18; allowing said labelled antibodies to bind to said complexes; and detecting said labelled antibody bound to said complexes.

12. The method according to claim 1, wherein said labelled antibodies are labelled with horse radish peroxidase and detection is performed by adding a substrate capable of being converted to a detectable substance by said horse radish peroxidase.

13. The method according to claim 12, wherein said substrate is converted to a chromogenic substance by said horse radish peroxidase.

14. The method according to claim 13, wherein said substrate is TMB (3,3,5,5-Tetramethylbenzidine).

15. The method for quantitative determination of soluble fragments of at least two cytokeratins selected from the group consisting of cytokeratin 8, 18 and 19 in a fluid sample of a human subject, comprising the method according to claim 1, and further comprising the step of: quantitatively correlating the amount of bound first labelled antibody having specificity for a dimer of cytokeratin 8 and 18 and, if present, second labelled antibody having specificity for cytokeratin 19 with known amounts of soluble fragments of cytokeratins 8, 18 and/or 19.

16. The method for quantitative determination of soluble fragments of at least two cytokeratins selected from the group consisting of cytokeratin 8, 18 and 19 in a fluid sample of a human subject, comprising the method according to claim 11, and further comprising the step of: quantitatively correlating the amount of bound first labelled antibody having specificity for a dimer of cytokeratin 8 and 18 and, if present, second labelled antibody having specificity for cytokeratin 19 with known amounts of soluble fragments of cytokeratins 8, 18 and/or 19.

17. A method for detecting at least two cytokeratins selected from the group consisting of cytokeratin 8, 18 and 19 and soluble fragments thereof in a fluid sample of a human subject, comprising the steps of contacting said fluid sample with a solid phase having immobilized thereon a first antibody having specificity for cytokeratin 8, a second antibody having specificity for cytokeratin 18 and optionally a third antibody having specificity for a first epitope of cytokeratin 19; allowing cytokeratins and soluble fragments thereof to bind to said first, second and optionally third antibodies thereby forming complexes; contacting said complexes with a first labelled antibody having specificity for a dimer of cytokeratin 8 and 18 and optionally a second labelled antibody having specificity for a second epitope of cytokeratin 19; allowing said labelled antibodies to bind to said complexes; and detecting said labelled antibodies bound to said complexes.

18. The method according to claim 17, wherein said antibodies are monoclonal antibodies.

19. The method according to claim 17, wherein said first labelled antibody has specificity for the -helix 2B 2 (aa 414-429 as set forth in SEQ ID NO:1) of cytokeratin 18, and, if present, said second labelled antibody has specificity for the -helix 2B 2 (aa 311-335 as set forth in SEQ ID NO:2) of cytokeratin 19.

20. The method according to claim 19, wherein said first labelled antibody is M21, and, if present, said second labelled antibody is A53-B/A2.

21. The method according to claim 17, wherein said first immobilized antibody has specificity for the -helix 2B (aa 340-365 as set forth in SEQ ID NO:3), said second immobilized antibody has specificity for the -helix 2B (aa 320-350 as set forth in SEQ ID NO:4), and, if present, said third immobilized antibody has specificity for the -helix 2B (aa 340-370 as set forth in SEQ ID NO:5).

22. The method according to claim 21, wherein said first immobilized antibody is 6D7, said second immobilized antibody is 3F3, and, if present, said third immobilized antibody is IDLC4.

23. The method according to claim 17, wherein said first antibody having specificity for cytokeratin 8 constitutes 20-40% of total immobilized antibody, said second antibody having specificity for cytokeratin 18, constitutes 5-15% of total immobilized antibody, and said third antibody having specificity for cytokeratin 19 constitutes 50-70% of total immobilized antibody.

24. The method according to claim 23, wherein said first antibody having specificity for cytokeratin 8 constitutes 30% of total immobilized antibody, said second antibody having specificity for cytokeratin 18, constitutes 10% of total immobilized antibody, and said third antibody having specificity for cytokeratin 19 constitutes 60% of total immobilized antibody.

25. The method according to claim 17, wherein the cytokeratin 8 and 18 and soluble fragments thereof in the fluid sample are detected by a method comprising the steps of: contacting said fluid sample with a solid phase having immobilized thereon a first antibody having specificity for cytokeratin 8, and a second antibody having specificity for cytokeratin 18; allowing cytokeratins 8 and 18 and soluble fragments thereof in said sample to bind to said first and second antibodies thereby forming complexes; contacting said complexes with a first labelled antibody having specificity for a dimer of cytokeratin 8 and 18; allowing said labelled antibodies to bind to said complexes; and detecting said labelled antibody bound to said complexes.

26. The method according to claim 17, wherein said labelled antibodies are labelled with horse radish peroxidase and detection is performed by adding a substrate capable of being converted to a detectable substance by said horse radish peroxidase.

27. The method according to claim 26, wherein said substrate is converted to a chromogenic substance by said horse radish peroxidase.

28. The method according to claim 27, wherein said substrate is TMB (3,3,5,5-Tetramethylbenzidine).

29. A method for quantitative determination of at least two cytokeratins selected from the group consisting of cytokeratin 8, 18 and 19 and soluble fragments thereof in a fluid sample of a human subject, comprising the method according to claim 17, and further comprising the step of: quantitatively correlating the amount of bound first labelled antibody having specificity for a dimer of cytokeratin 8 and 18 and, if present, second labelled antibody having specificity for cytokeratin 19 with known amounts of soluble fragments of cytokeratins 8, 18 and/or 19.

30. A method for quantitative determination of at least two cytokeratins selected from the group consisting of cytokeratin 8, 18 and 19 and soluble fragments thereof in a fluid sample of a human subject, comprising the method according to claim 25, and further comprising the step of: quantitatively correlating the amount of bound first labelled antibody having specificity for a dimer of cytokeratin 8 and 18 and, if present, second labelled antibody having specificity for cytokeratin 19 with known amounts of soluble fragments of cytokeratins 8, 18 and/or 19.

Description

BRIEF DESCRIPTION OF THE APPENDED DRAWINGS

[0023] FIG. 1 is a schematic overview of how to perform the method according to the present invention.

[0024] FIGS. 2A-2D show the results obtained in example 2.1.

[0025] FIG. 3 is a diagram showing the results obtained in example 2.2.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

[0026] The present inventors have identified a need to produce new or find existing antibodies to facilitate an assay without the drawbacks of prior art technology, while retaining a good clinical correlation with the existing assay, MonoTotal IRMA. In the detailed description below, the term having affinity for is used for antibodies which are specific for their respective targets, as will easily be recognized from the context by the skilled person.

[0027] A new assay for sensitive indication of tumour cell activity useful in the management of patients with carcinomas of epithelial origin, especially non-small cell lung carcinoma was therefore developed by the present inventors. An enzyme-linked immunosorbent assay (ELISA) format was chosen herein, but the skilled person will recognize that alternative formats utilizing the principles of the method according to the invention are also within the scope of the present invention.

[0028] In one embodiment of the present invention, where the three cytokeratines 8, 18 and 19 are detected, a requirement for use of monoclonal antibodies is maintained specificity for cytokeratin 19 as compared to the existing immunoradiometric assay and also good clinical correlation with the existing assay. The assay must also be stable and have acceptable signal strength.

[0029] This embodiment is advantageous in that the detection of cytokeratin 19 has been shown to be clinically superior in NSCLC. To increase sensitivity and taking into account that cytokeratins form heterodimers and that certain antibody epitopes only are available on the dimeric form, an assay was constructed for detection of cytokeratins 8, 18 and 19.

[0030] Surprisingly, a very good clinical correlation with the prior art assay could be achieved in this embodiment by combining a first labelled antibody with affinity for a dimer of cytokeratin 8 and 18 with a second labelled antibody with affinity for cytokeratin 19. The first labelled antibody should show substantially no affinity to monomers or homodimers of cytokeratin 8 or 18.

[0031] Thus, this embodiment of the method of the invention utilizes a first, second and third antibody bound to a solid phase, such as a standard microtitre plate. The first, second and third antibody immobilized to the solid phase may be produced as disclosed e.g. in WO92/05197, using purified fragments of cytokeratin 8, 18 and 19, respectively. An antibody denoted 6D7, specific for cytokeratin 8, and an antibody denoted 3F3, specific for cytokeratin 18, have been produced by this method as described in Siln et al, Scand J Clin Invest, 1995, 55, 153-161 and Stigbrand et al., Tumor Biol 1998, 19, 132-152. The antibody IDLC4 is specific for an epitope of cytokeratin 19 in the region of amino acid residues 340-370 (as set forth in SEQ ID NO:5), as discussed in Brattstrm et al., Diseases of the esophagus, 2005, 18, 298-303.

[0032] The first labelled antibody having an affinity for a dimer of cytokeratin 8 and 18 may be produced with the method according to Rydlander et al., Eur J Biochem, 1996, 241, 309-315 and screened for affinity to a dimer of cytokeratin 8 and 18 and lack of affinity to monomeric forms of cytokeratin 8 and 18. This is further explained in Example 2 below, where a suitable antibody named M21 is produced. Alternatively, the first labelled antibody may be a commercially available antibody such as A45-B/B3 (Biologicals Ltd., U.K.) or DE-K18 (Fisher Scientific). The cell line producing 6D7 has been deposited with the European Collection of Cell Cultures (ECACC, Porton Down, United Kingdom) on 19 Mar. 2013 under accession number 13031902. The cell line producing 3F3 6D7 has been deposited with the ECACC on 19 Mar. 2013 under accession number 13031901. The cell line producing IDLC4 has been deposited with the ECACC on 19 Mar. 2013 under accession number 13031903. The cell line producing M21 has been deposited with the ECACC on 21 Mar. 2013 under accession number 13032101 The second labelled antibody used in this embodiment of the method according to the invention having affinity for cytokeratin 19 may be any antibody with affinity to cytokeratin 19 such as a commercially available antibody, e.g. A53-B/A2 (Ventana, Tucson Ariz., U.S.A.).

[0033] In another embodiment of the method according to the invention cytokeratin 8 and 18, and/or soluble fragments thereof, in a sample are detected. More specifically, this embodiment of the method comprises the steps of [0034] contacting said sample with a solid phase having immobilized thereon a first antibody having specificity for cytokeratin 8, and a second antibody having specificity for cytokeratin 18; [0035] allowing cytokeratin and/or soluble fragments thereof in said sample to bind to said first and second antibodies thereby forming complexes; [0036] contacting said complexes with a first labelled antibody having specificity for a dimer of cytokeratin 8 and 18; [0037] allowing said labelled antibodies to bind to said complexes; and [0038] detecting said labelled antibody bound to said complexes.

[0039] The details above relating e.g. to antibodies, their manufacture etc of the first embodiment are also applicable to this embodiment.

[0040] The above-described two embodiments, and other embodiments of the method according to the invention, are advantageously performed as one step enzyme-linked sandwich immunoassays (ELISAs). Standards, controls and samples react during incubation simultaneously with monoclonal catcher antibodies immobilized on a solid phase and Horse Radish Peroxidase (HRP)-conjugated detector antibodies. After washing, a developer substrate, e.g. 3,3,5,5-Tetramethylbenzidine (TMB), is added and after an incubation time the reaction is stopped and the absorbance at 450 nm is measured. The developed color is directly proportional to the concentration of the analyte.

[0041] In one embodiment, an assay according to the present invention is performed with serum samples. Enough blood should be collected to be sufficient for 2100 l serum (duplicates) at each analysis. If the analysis is performed within 24 h, the serum should be stored refrigerated (2-8 C.). If delayed analysis, the serum is preferably stored frozen <18 C. Repeated thawing and freezing should be avoided. Serum samples that are highly hemolysed, grossly lipemic or contaminated should not be used.

[0042] The assay procedure is schematically shown in FIG. 1. [0043] 1. Pipette 100 l standards, controls or samples per defined well. Leave two empty wells (blank) for background absorbance measurement (optional). [0044] 2. Add 100 l first and second labelled antibody in each well except the blank wells. [0045] 3. Incubate for 2 h+10 min on a shaker at 450 rpm. [0046] 4. Aspirate and wash the wells 3 times with 0.3 ml wash solution. [0047] 5. Add 200 l TMB substrate per well, including the blank wells. [0048] 6. Incubate in darkness for 151 min. [0049] 7. Add 100 l Stop Solution per well. Agitate on a shaker for 1 min. [0050] 8. Read the absorbance at 450 nm within 30 min after addition of the Stop Solution. Calculate the cytokeratin 8/18/19 concentration of the samples. Samples should be diluted as appropriate, as easily determined by the skilled person, with diluent before repeated analysis.

[0051] The absorbance at 450 nm is read using a standard microplate reader (wavelength 450 nm). Computer software is used for handling the raw data. Spline smoothed is recommended as a curve fitting algorithm. For generation of valid data, it should be ensured that included controls are within range.

[0052] Manual processing of results: Correct each OD-value (optical density) by subtracting the blank OD. Calculate the mean OD-value for each duplicate. Construct a standard curve by plotting the mean OD-value for each standard (y-axis) against the corresponding concentration (x-axis). Determine the concentrations of the samples against the standard curve.

[0053] In an alternative embodiment, the method according to the present invention is performed on a urine sample in order to detect bladder cancer. Based on the information in this application and common general knowledge, the skilled person is able to prepare such a sample in the appropriate way for the detection according to the invention.

[0054] The invention further relates to a kit of parts for performing the method described above. Such a kit may advantageously comprise [0055] a solid phase having immobilized thereon a first antibody having affinity for cytokeratin 8, and/or a second antibody having affinity for cytokeratin, 18 and/or a third antibody having affinity for cytokeratin 19; and [0056] a first labelled antibody having affinity for a dimer of cytokeratin 8 and 18 and/or a second labelled antibody having affinity for cytokeratin 19.

[0057] The labelled antibodies are preferably provided in one or more buffered solutions. The labelled antibodies are in one embodiment labelled by conjugation to Horse Radish Peroxidase.

[0058] The kit may also comprise means for diluting samples, e.g. a ready made protein stabilized buffer of pH 7.5 which may or may not further comprise preservatives, or a dry mix for preparing such buffer when mixed with water.

[0059] The kit may further comprise means for use as standards, e.g. standard materials in protein stabilized buffer or a dry mix for preparing such buffer when mixed with water. The standards may be provided in several different concentrations.

[0060] The kit may further comprise means for use as controls, e.g. control materials in protein stabilized buffer or a dry mix for preparing such buffer when mixed with water. The standards may be provided in several different concentrations, e.g. low and high.

[0061] The kit may further comprise means for washing the solid phase (cf. step 4 in FIG. 1). The washing means may be provided as a ready-made washing solution or as a wash tablet for dissolving in water.

[0062] The kit according to the invention may also comprise written instructions for the detection of lung cancer, preferably non-small cell lung cancer; or for the detection of bladder cancer.

[0063] Although the presently preferred format for the assay according to the invention is Enzyme-Linked ImmunoSorbent Assay (ELISA), other immunoassay principles may be used to put the invention to practice. Such immunoassay principles include i.a. radioimmunoassays, fluorescence immunoassays, luminiscent oxygen channeling assay (LOCI), Surface Plasmon Resonance (SPR), ellipsometry, luminescence immunoassay (luciferase/ATP), chemiluminiscence immunoassays (dioxetane, acridinium, acridinium esters, luminol, isoluminol), electrochemiluminiscence immunoassay (ruthenium salts), dissociation enhancement time-resolved fluoroimmunoassay (DELFIA), electrical detection (conductance, carbon nanotube, semiconductor nanowires, silicone-nanowires), and Luminex (laser fluorophore).

[0064] The present invention is not limited to the above-described preferred embodiments. Various alternatives, modifications and equivalents may be used. Therefore, the above embodiments or the illustrative examples below should not be taken as limiting the scope of the invention, which is defined by the appended claims.

[0065] When practicing the present invention the person skilled in the art may further make of use conventional techniques in the field of pharmaceutical chemistry, immunology, molecular biology, microbiology, cell biology, and recombinant DNA technology, as i.a. disclosed in Sambrook et al. Molecular cloning: A laboratory manual, 3.sup.rd ed. 2001; Ausubel et al. Short protocols in molecular biology, 5.sup.th ed. 1995; Methods in enzymology, Academic Press, Inc.; MacPherson, Hames and Taylor (eds.). PCR 2: A practical approach, 1995; Harlow and Lane (eds.) Antibodies, a laboratory manual 1988; Freshney (ed.) Culture of animal cells, 4.sup.th ed. 2000; Methods in Molecular Biology vol. 149 (The ELISA Guidebook by John Crowther) Humana Press 2001, or later editions of these books.

EXPERIMENTAL PART

[0066] The examples below are provided for illustrative purposes only, and should not be construed as limiting the invention which is defined by the appended claims. All references provided below and elsewhere in the present application are hereby included herein by reference.

Example 1

[0067] In order to find a suitable monoclonal detector antibody for conjugation in the ELISA format a number of monoclonal antibodies were evaluated. Interaction between cytokeratins 8, 18 and 19 bound to catcher antibodies (3F3, 6D7 and IDLC4) were evaluated in real time using a Quartz Crystal Microbalance biosensor instrument from Attana AB (Stockholm, Sweden), providing information on binding strength, on-rate, binding level and binding stability and epitope similarity.

[0068] Six antibodies with affinity for cytokeratin 19, three with affinity for cytokeratin 8, four with affinity for cytokeratin 18 and one with affinity for a number of cytokeratins were evaluated, cf.

[0069] Table 1.

TABLE-US-00001 TABLE 1 Anti CK 19 Anti CK 8 Anti CK 18 Pan ab IDL C3 IDL33 3F3 C 11 IDL C4 6D7 M3 IDL C5 2D1 M21 A53-B/A2 M34 BA 17 HCA 077

[0070] Based on results from the biosensor tests, antibodies A53-B/A2, IDL33, HCA 077, M21 and C11 were conjugated to HRP and tested in enzyme-linked immunosorbent assays and evaluated for clinical correlation with the prior art assay.

[0071] 12 samples or pooled samples from cancer patients diluted 1:2 or 1:4 in MonoTotal diluent were used for comparison. The conjugated antibodies or mixes of antibodies were diluted to a final concentration of 1 g/ml. A standard of 5472 U/1 diluted 1:2 in six steps were used.

[0072] Protocol:

[0073] 100 l standard/sample/diluted sample

[0074] 100 l conjugated antibody

[0075] 2 hours shaking 450 rpm

[0076] Wash 2*3*300 l

[0077] 200 l TMB

[0078] 15 min incubation in the dark

[0079] 100 l stop solution

[0080] 1 min shaking

[0081] Reader: 450 nm.

[0082] Results

[0083] The concentrations for the various assay variants were generated in Gen5 and plotted versus MonoTotal IRMA data, whereafter a linear fit was made in MS Excel. The results are summarised in Table 2.

TABLE-US-00002 TABLE 2 Conjugate R.sup.2 C11 0.8023 M21 0.9231 A53-B/A2 0.9395 HCA 077 0.8435 C11 + M21 0.9357 C11 + A53-B/A2 0.9579 C11 + HCA 077 0.9339 M21 + A53-B/A2 0.9936 M21 + HCA 077 0.8615 A53-B/A2 + HCA 077 0.9479

[0084] Surprisingly, a very good clinical correlation (R.sup.2=0.9936) with the prior art assay could be achieved by combining a monoclonal antibody (M21) with affinity for cytokeratin 18 but without known affinity for cytokeratin 8, with a monoclonal antibody with affinity for cytokeratin 19 (A53-B/A2).

Example 2

[0085] This example shows that the antibody M21 as used in Example 1 binds to a heterodimer of cytokeratins 8 and 18, but not to pure cytokeratin 18. M21 has previously been shown to bind to cytokeratin 18, see Rydlander et al, Eur. J. Biochem. 1996, 241, 3009-314, but it has not been shown that this binding requires that the cytokeratin 18 is bound to cytokeratin 8.

Example 2.1

[0086] Avidin strips were coated with biotin coupled antibodies M21, M34 and 3F3 in room temperature and washed with 3*300 l wash solution. The coated strips were incubated with a standard solution of culture supernatant from a tumor cell line containing cytokeratin fragments from cytoskeleton, mainly cytokeratins 8, 18 and 19, in concentrations 0, 120 and 1200 U/I or a reference solution of pure recombinant cytokeratin 18 in dilutions 1:1, 1:2, 1:4, 1:8. Bound cytokeratin 18 were detected by HRP-conjugated monoclonal antibody M3 or polyclonal horse anti-TPS antibody P03.

[0087] The results are summarized in Table 3 and shown in FIG. 2A-D.

TABLE-US-00003 TABLE 3 HRP-conjugated M3 HRP-conjugated P03 M34 3F3 M21 M34 3F3 M21 Blank 0.059 0.058 0.056 Blank 0.062 0.059 0.056 Std 0 0.057 0.059 0.056 Std 0 0.139 0.133 1.172 Std 120 0.058 0.061 0.19 Std 120 0.152 0.265 0.344 Std 1200 0.098 0.065 1.343 Std 1200 0.181 1.451 1.047 CK18 ref 0.828 0.058 0.062 CK18 ref 0.637 0.717 0.218 1:2 0.411 0.063 0.072 1:2 0.341 0.375 0.259 1:4 0.227 0.059 0.062 1:4 0.239 0.233 0.199 1:8 0.145 0.066 0.065 1:8 0.184 0.197 0.173

[0088] The results clearly indicate that the antibody M21 essentially lack affinity for pure recombinant cytokeratin 18, but has affinity to a mixture of cytokeratins 8, 18 and 19.

Example 2.2

[0089] Stock solutions of antibody M21 (0.65 mg/ml) and M34 (0.58 mg/ml) were prepared.

[0090] The wells of a 96-well microtitre plate for ELISA were coated with M21 antibody and a mixture of M21 and M34 antibody in six different variants:

[0091] Reference: TPS standard (polyclonal horse anti-TPS)

[0092] Variant 1: 12 g/ml M21+0.5 mg/ml Bovine Serum Albumin

[0093] Variant 2: 16 g/ml M21+0.5 mg/ml BSA

[0094] Variant 3:20 g/ml M21+0.5 mg/ml BSA

[0095] Variant 4: 12 g/ml M21+4 g/ml M34

[0096] Variant 5: 16 g/ml M21+4 g/ml M34

[0097] Variant 6: 20 g/ml M21+4 g/ml M34

[0098] To each variant was added 50 l cancer sample (pooled samples from Breast Cancer (BC) patients), normal sample (from healthy Blood Donors (BD)), TPS (tissue-polypeptide specific antigen) diluent (0 U/l) or TPS standard (1200 U/l) (IDL Biotech, Bromma, Sweden), recombinant cytokeratin 18, or a mixture of recombinant cytokeratin 8 and 18. 50 l HRP-conjugated mAb M3 200 ng/ml was added, the wells were incubated on a shaker in room temperature for 2 hours at 450 rpm and then washed three times with 300 l wash solution. 200 l TMB substrate was added and the wells were incubated 20 minutes in the dark. 50 l stop solution was added and the wells were shaken for 1 minute. Bound cytokeratins was detected by measuring the absorbance at 450 nm.

[0099] The results are shown in Table 4 and FIG. 3.

TABLE-US-00004 TABLE 4 Reference Variant1 Variant2 Variant3 Variant4 Variant5 Variant6 Diluent 0.089 0.082 0.087 0.084 0.084 0.087 0.088 TPS std 1.736 1.218 1.201 1.307 1.109 1.243 1.341 CK8/CK18 0.839 0.671 0.71 0.717 0.636 0.669 0.681 CK18 1.317 0.086 0.084 0.088 0.299 0.301 0.259 TPAref 0.793 0.513 0.494 0.544 0.48 0.536 0.66 BD sample 0.22 0.14 0.141 0.152 0.141 0.15 0.147 BC sample 0.749 0.41 0.391 0.404 0.391 0.397 0.402

[0100] The results show that M21 alone shows affinity for the combination of cytokeratins 8 and 18, but not for pure cytokeratin 18.

[0101] Analogous experiments will show that M21 does not show affinity for pure cytokeratin 8, i.e. cytokeratin 8 in the absence of cytokeratin 18.

Example 3

[0102] This example shows that an advantageous assay format may be provided by replacing the polyclonal antibody from horse used in the prior art (UBC/TPAcyk) with two monoclonal antibodies, which are specific to cytokeratin 8 (CK8) and cytokeratin 18 (CK18).

[0103] The antibodies were first tested for epitope similarity and kinetics in an instrument from Attana A100 QCM Research System (website attana.com). The test was performed on polystyrene chips coated with antibody 6D7/3F3 in proportions equivalent to an ELISA test. The antibodies that worked satisfactorily were conjugated and for further tests in ELISA.

[0104] The conjugates in the table below are of the concentration 1 g/ml. The binding (ABS) of the highest standard is reported.

TABLE-US-00005 Ab Specificity Attana result UBC ELISA IDL33 CK 8 Very low binding UBC std. 15 g/l = 0.25 2D1 CK 8 High binding UBC High off-rate std. 15 g/l = 0.17 M21 CK 18 High binding UBC Higher on-rate std. 15 g/l = 0.73 and lower off-rate UBC compared to 2D1 std. 15 g/l = 0.64 Binding is partly Std1 < blg < std 2 inhibited by 2D1 UBC std. 15 g/l = 0.65 M3 CK 18 Medium binding UBC Low off-rate std. 15 g/l = 0.52 Binding is partly inhibited by all antibodies except 2D1 C11 Pan ak High binding UBC Bl.a CK 8 Slower on-rate std. 15 g/l = 1.37 and 18 compared to M21 UBC Low off-rate std. 15 g/l = 1.48 No inhibition from Std0 < blg < std 1 the other antibodies UBC std. 15 g/l = 1.50

[0105] UBC EISA with Different Conjugates

[0106] The plate was coated with equal parts of 6D7 (anti CK8) and 3F3 (anti CK18), the standard is comprised of equal parts of recombinant CK8 and CK18. Tests were performed with internal controls and patient samples.

TABLE-US-00006 Binding to std. 0 Correlation Correlation Conjugate and 15 to the prior coefficient Number of (1 g/ml) g/l (ABS) art assay (r.sup.2) samples C11 0.050/1.5 y = 6.7x 0.1 0.80 27 C11 + M21 0.060/1.6 y = 1.2x + 0.2 0.92 27 M21 0.065/0.7 y = 1.3x + 0.8 0.91 12 C11 + M21 0.055/1.9 y = 1.6x + 1 0.94 12

[0107] As appears from above, C11 works well, it bound after all antibodies investigated, and has an epitope on both CK8 and CK18.

[0108] M21 alone gives a good correlation to the prior art assay but very low binding.

[0109] The combination of C11+M21 provides a binding which is roughly equivalent to C11 alone. The combination provides good binding and good correlation. The combination also allows a lower concentration of C11, and may be an advantageous economic alternative.

CONCLUSION

[0110] This example shows that the combination of C11 with M21 is a fully operable alternative to the prior art assay.