WO2008119882A2

WO2008119882A2 - Screening methods based on human adam12 polypeptide and binding agents thereof

Info

Publication number: WO2008119882A2
Application number: PCT/FI2008/050146
Authority: WO
Inventors: Mikko SUONPÄÄ; Päivi ARONKYTÖ; Pertti Hurskainen; Outi Mulari; Jarkko Karvinen
Original assignee: Wallac Oy
Current assignee: Wallac Oy
Priority date: 2007-03-30
Filing date: 2008-03-28
Publication date: 2008-10-09
Anticipated expiration: 2009-09-30
Also published as: WO2008119882A8; WO2008119882A3

Abstract

The present invention relates to a novel epitope of human ADAM12 and to binding agents, preferably to Fab fragments, specifically binding to said epitope. The invention also relates to a method for detecting ADAM 12 in a biological sample using said binding agents, and to prenatal screening methods and diagnostic methods using said binding agents.

Description

SCREENING METHODS

FIELD OF THE INVENTION

The present invention relates to binding agents, preferably monoclonal antibody FAb fragments recognizing novel epitopes of ADAM12, useful for e.g. screening Down's syndrome in samples taken during the pregnancy.

BACKGROUND OF THE INVENTION

Human ADAM12, an acronyme for A Disintegrin And Metalloprote- ase 12 is glycoprotein, which belongs to metzincin superfamily. It is synthe- sised in two forms ADAM12-L a 909 amino acid cell membrane bound form and ADAM12-S (hereinafter ADAM12), a 739 amino acid secreted form.

ADAM 12 is synthesized by the placenta and secreted throughout the pregnancy. ADAM 12 is a potentially important factor to screen for in prenatal diagnosis of Down syndrome during the first trimester, as the level of ADAM12 is reduced in maternal serum during the first trimester in Down syndrome and trisomy 18 pregnancies, as described by Laigaard et al, in Prenatal Diagnosis, 26 (2006). Furthermore, ADAM12 is reduced in the serum of women later developing preeclampsia. US patent publications 2005/025114 and 2006/0134654 disclose use of ADAM 12 as a marker in antibody based assays for diagnosing fetal chromosomal abnormalities and preeclampsia. In addition to placenta, many cancer cells express immunoreactive

ADAM 12 (Iba et al., (1999) Amer J Pathology, 154, 1489-1500). ADAM12 has also been found in the urine of breast cancer patients. International patent publication WO 2005/071387 discloses ADAM12 as a marker in antibody based assays for diagnosing cancers of epithelial origin and providing a prog- nosis thereof.

Monoclonal antibodies against recombinant ADAM 12 have been disclosed and Laigaard et al. 2006 describe a semi-automated time-resolved immunofluorometric assay using the AutoDELFIA platform (PerkinElmer, Turku, Finland). In this assay, recombinant human ADAM12 was used for standardization, and a previously described MAb (6E6) disclosed by Gilpin et al. (1998) was coated onto the surface of microtiter plates, and a biotinylated MAb (8F8) was used for detection. Furthermore, epitopes of ADAM12 and monoclonal antibodies recognizing the epitopes have been disclosed in US patent publications 2006/0247164 and 200&014903. ^{" "} However, it is here shown that ADAM12 undergoes structural changes in serum samples upon storage in a temperature dependent manner, making the above test method questionable. There is thus a need for an improved assay and improved reagents.

BRIEF DESCRIPTION OF THE INVENTION

The present invention related to a novel, isolated epitope of human ADAM12, which is found in SEQ ID NO: 2, i.e., amino acids 360-660. Preferably, the epitope comprises the amino acid sequence SEQ ID NO: 3 and at least one amino acid sequence selected from the group consisting of:; SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.

The invention further relates to a binding agent that specifically binds to an epitope of ADAM12 according to the present invention. Preferably the binding agent according to the present invention is a polyclonal or monoclonal antibody or a fragment thereof. The invention also relates to a method for detecting ADAM 12 in a biological sample, wherein a sample, preferably a serum sample, obtained from a patient is provided and said sample is contacted with a binding agent according to the present invention and with another binding agent, preferably with the monoclonal antibodies, such as mAb 6E6, capable of binding ADAM12. ADAM12 is detected by means of a label, either in one of the binding reagent itself or in the antigen. Preferably, one of the binding agents is immobilized on a solid support and the other binding agent is labeled. The intensity of the signal produced by the bound labeled binding agent is proportional to the concentration of ADAM 12 in the sample. The invention further relates to a method for prenatal screening of conditions related to ADAM 12, wherein a serum sample obtained from a woman during pregnancy, preferably during the first trimester, is provided and contacted with a binding agent according to the present invention. The level of ADAM 12 in said serum sample is determined and compared to median levels of ADAM12 in serum. An abnormal, e.g., a decreased, level of ADAM12 is indicative of said condition. Preferably said conditions to be screened are fetal chromosomal anomalies e.g, Down's syndrome and trisomy 18, or preeclampsia, intrauterine growth retardation, fetal death, pre-term birth and low birth weight. The invention further relates to a method for diagnosing other diseases where ADAM12 is abnormal e.g. cancers preferably breast cancer. In such cases the level of ADAM12 may be elevated.

Finally, the present invention relates to a kit for detecting ADAM 12, comprising a binding agent according to the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following the invention will be described in greater detail by means of preferred embodiments with reference to the figures, in which

Figure 1 is a graph illustrating the instability of ADAM12 in serum samples when detected by monoclonal antibody 8F8, and

Figure 2 is a graph showing the sample stability in respect of ADAM12 when using Fab fragment FAb52.

DETAILED DESCRIPTION OF THE INVENTION

A sandwich immunoassay based on two ADAM12-specific mono- clonal antibodies such as the ones described by Laigaard et al, could be useful for the detection of human ADAM12 in serum samples. In such an assay, one monoclonal antibody, such as clone 6E6 described by Laigaard et al, is used as a capture and is coated on a solid support, such as a microtitration plate, while another monoclonal antibody, such as clone 8F8 described by Laigaard et al., is labelled for detection purposes, with e.g., DELFIA Eu chelate.

When further developing and testing such an assay, it was surprisingly noted that the ADAM 12 concentrations in maternal serum samples decreased markedly upon storage. Serum samples stored at +4°C for several days showed up to a 30% decrease in ADAM12 concentrations. Results were even more troublesome when serum samples were stored at room temperature (21⁰C), where the ADAM12 concentration showed a 60-70% decrease after storage of 7 days. As clinical samples taken at local hospitals or healthcare centers often are sent to central hospitals or laboratories for testing, either by mail or courier service, lability of ADAM 12 is a notable problem. The tem- perature during transportation of the samples may vary and can be about +20°C or higher for several days. An assay based on two monoclonal antibodies, as suggested in the art, gives ADAM 12 concentrations which are largely dependent on the conditions during sample transportation. This fact makes such an assay unreliable and consequently unsuitable for diagnosis of dis- eases or prenatal screening of Down's syndrome in the clinics.^' The known antibodies against ADAM 12, especially 8F8 seems to be sensitive to conformation changes in the target they have been raised against.

Thus, the present inventors set out to develop improved binding agents for use in immunoassay for detecting ADAM 12. Such reagents should not be as sensi- tive to the possible conformation changes in the target, ADAM12.

It is an object of the present invention to provide improved binding agents for detecting ADAM12 in e.g. serum samples, which agents are less sensitive to the temperature dependent conformation changes of ADAM 12. Suitable agents are any compound binding specifically to ADAM12 in a posi- tion which is not as sensitive to storage conditions of the target as prior art antibodies. Examples of such binding agents include polyclonal or monoclonal antibodies and fragments thereof, binding peptides, and aptamers. Preferably, binding agents useful in the present invention, recognize different epitopes of ADAM 12 as compared to MAb 8F8. One potential approach for providing reagents for detecting

ADAM 12 according to the present invention is to provide antibody fragments, such as a Fab, a Fv or a scFv fragment, especially Fab fragments. Such fragments are highly preferred binding agents due to their smaller size and easy of production. Fab fragments are usually more stable than Fv fragments, and were thus chosen as a working hypothesis when searching for improved ADAM12 binding agents.

Fab fragments are monovalent and consist of a heavy chain variable region and a light chain variable region. When so-called association domains are fused to C-terminus of a heavy chain variable region (for a review see Pluckthun and Pack, 1997) dimeric Fab fragments are formed. Recombinant bivalent Fab fragments contain four self-associating domains (two per monomeric Fab fragment separated by a short flexible spacer sequence) which form a four-helix bundle. All four helices have the same amino acid sequence. Additionally peptide tags, such as 6xHis, StrepTag and Flag, may be incorporated to C-terminus for purification and possibly also for detection purposes. Avidity of these dimeric Fab fragments is about ten times higher than avidity of corresponding monomeric Fab fragments.

Antibody fragments may be produced by phage display methods. This concept was originally described for producing short peptides by Smith in 1985, and for producing functional antibody fragments in US Patent No. 6,248,416. A major advantage of phage display technology for producing Fab fragments is the ease and speed by which a large number of antigen specific fragments can be screened.

The starting point for selecting specific Fab fragments today is often a large antibody library comprising billions of clones. There are several com- mercially available libraries, and several companies provide screening service, wherein a target antigen is screened against the vast phage display library. After two to three rounds of selection an enriched population of antibody fragments specific for the antigen is provided.

Purified recombinant mature, full-length form of ADAM12 (GenBank accession number AF023477; SEQ ID NO. 1 ) was screened by panning against a human, highly variable monomeric Fab fragment library (Human Combinatorial Antibody Library, HuCAL; AbD Serotec) consisting of up to billions of Fab fragments in a phage library. Positive clones were identified as potentially antigen specific Fab fragments using ELISA where the fragments were reacted with recombinant ADAM12 coated onto a solid support. Bound Fab fragments were detected by enzyme labelled streptavidin.

The positive clones may be converted into clones producing dimeric Fab fragments to increase Fab avidity, expressed in bacteria, such as E.coli and purified. The purification step may be performed by introducing pep- tide tags, such as StrepTag described by Skerra et al, in Biomolecular engineering, 16:79-86, 1999, into the Fab fragments, and using a streptavidin column for purification.

The best ADAM12 specific Fab fragments thus identified were further analyzed and epitope mapped, in order to identify novel epitopes of ADAM12, which are not sensitive to temperature dependent conformation change of ADAM 12 in serum samples. Fab fragments may be mapped using synthetic peptide scanning. Chemical spot synthesis of short peptides, for example 20-mer peptides, overlapping by 15, may be performed on cellulose membranes using Fmoc chemistry or alternatively peptide libraries can be used as described by Valadon et al, in I. Immonol. Methods, 197:171-179, 1996. Peptides are preferably synthesized so that they cover the entire target protein, i.e., human ADAM12.

Such a library e.g. membrane bound epitope library is then screened against Fab fragments raised against the target protein, ADAM 12. Such fragments are labelled for detection purpose, preferable biotinylated. After incubation and washing of the membranes, labelled Fab fragments are detected. The detection step may be performed by treating the membranes with horseradish peroxidase-conjugated streptavidin. Following washes the Fab fragments bound to the immobilized peptide epitopes are detected, e.g, as described in the examples, and corresponding peptide sequences are identi- fied as ADAM 12 epitopes.

Using such an approach, as described in the examples, several positive sequences in human ADAM 12 were identified. A novel epitope was identified in the amino acid stretch defined by amino acids 360-660 (SEQ ID NO: 2), comprising at least one of the amino acid stretches defined by amino acids 445-475 (SEQ ID NO: 3), amino acids 560-580 (SEQ ID NO: 4), amino acids 625-645 (SEQ ID NO: 5), amino acids 650-660 (SEQ ID NO: 6) and amino acids 360-380 (SEQ ID NO: 7) was thus identified. Amino acids 445-475 are in the disintegrin domain, amino acids 560-580, 625-645 and 650-660 are in the cysteine-rich domain, whereas amino acids 360-380 are located in the metalloprotease domain of human ADAM 12.

It is thus an object of the present invention to provide a novel epitope of human ADAM 12, comprising the amino acid sequence defined by amino acids 445-475 (SEQ ID NO: 3) and at least one amino acid sequence defined by amino acids 560-580 (SEQ ID NO: 4), amino acids 625-645 (SEQ ID NO: 5), amino acids 650-660 (SEQ ID NO: 6) and amino acids 360-380 (SEQ ID NO: 7).

Isolated immobilized epitopes according to the present invention may be used to screen for further binding agents, such as aptamers, which are ADAM12 specific and which detect ADAM12 in body fluids, such as serum, whole blood, blood spots, plasma, urine or amniotic fluid.

Human ADAM 12 specific dimeric Fab fragments identified by panning against a commercialiy available antibody library were tested in a sandwich immunoassay. The Fab fragments were labelled with DELFIA Eu-labeling reagent (PerkinElmer) and purified using gel filtration. The assay was per- formed in micro-titration plates coated with ADAM12-specific IgG class monoclonal antibody (6E6). Dilutions of recombinant ADAM 12 and serum samples together with Eu-labelled Fab fragment were added to the wells. Following incubation and washing Eu signal was developed with DELFlA Enhancement solution (PerkinElmer). Subsequently, it was noted, that two of the Eu-labelled Fab fragments, denoted Fab1 and Fab52, were clearly superior to the monoclonal anti- body 8F8 in respect of detecting ADAM 12 in stored maternal serum samples. The decrease in ADAM 12 concentration of serum samples after storage at 4°C and RT for up to 6 days was on the average only 5-15% (see Figure 2). Such an improvement renders a screening test for ADAM12 clinically feasible, as it allows samples to be delivered by mail or courier. Furthermore, samples stored at -2O⁰C are stable when Fab fragments are used as tracers in DELFIA ADAM12 assay. This makes it possible to run retrospective studies and analyze earlier collected samples stored at -20⁰C.

Antibody fragments, preferably Fab fragments, detecting the ADAM12 epitopes according to the present invention, are especially useful as binding agents in assays for determining ADAM12 concentrations in biological samples, due to their lower sensitivity to the temperature dependent conformation changes of ADAM 12 in samples during storage.

It is thus a further object of the present invention to provide ADAM 12 specific binding agents, such as antibody fragments, especially Fab fragments, which detect an epitope, comprising the amino acid sequence defined by amino acids 445-475 (SEQ ID NO: 3) and at least one amino acid sequence defined by amino acids 560-580 (SEQ ID NO: 4), amino acids 625-645 (SEQ ID NO: 5), amino acids 650-660 (SEQ ID NO: 6) and amino acids 360- 380 (SEQ ID NO: 7).

Binding agents, preferably Fab fragments, according to the present invention, are useful in assays for detecting ADAM12 in body fluid samples, such as serum, whole blood, blood spots, plasma, urine or amniotic fluid, and thus useful for e.g. prenatal diagnosis of Down's syndrome, trisomy 18 or preeclampsia, as they are not sensitive to the temperature dependent conformation changes of ADAM12, which is occurring in serum samples during storage.

It is thus an object of the present invention to provide clinically feasible immunoassays for detecting ADAM 12 in maternal serum samples, using ADAM 12 specific binding agents, such as Fab fragments, which identify an epitope according to the present invention. An especially useful assay according to the present invention is a solid phase, two-site fluoroimmunometric assay based on the direct sandwich technique in which two binding agents, such as ADAM12-specific antibodies, or fragments thereof, are directed against two separate antigenic determinants on the ADAM12 molecule. Calibrators, controls and samples containing ADAM 12, are reacted with a first, immobilized ADAM 12 specific monoclonal antibody, such as 6E6. Bound ADAM 12 is detected with a second, labeled ADAM12-specific binding agent, such as a europium-labelled Fab fragment. Enhancement Solution dissociates europium ions from the labelled antibodies into the solution where they form highly fluores- cent chelates with components of the Enhancement Solution. The fluorescence in each well is then measured. The europium fluorescence of each sample is proportional to the concentration of ADAM 12 in the sample.

In specific embodiments according to the present invention said immobilizaed binding agent may be immobilized to any suitable solid support, such as reaction vials, microtitration plates, beads or slides.

In further, specific embodiments according to the present invention said detection may be utilizing suitable labels known in the art, such as fluorescent labels, lanthanide chelates, enzymes, luminescent labels, chemilumi- nescent labels, phosphorescent labels or radioisotopes. In another embodiment of the present invention, there is provided a homogeneous assay for detecting ADAM12. The detection of ADAM12 present in the sample to be tested may, in such an assay, be detected by TR- FRET or any other labeling system suitable for homogeneous assays known in the art. The present invention further provides a kit for use in an assay according to the present invention, comprising ADAM12 specific binding agents according to the present invention, ADAM12 calibrators, assay buffers, reaction vials coated with an ADAM12 specific binding agent recognizing a second antigenic determinanta of ADAM 12. Preferably, the binding agents according to the present invention included in said kit are labeled for detection purposes, preferably by a Eu-label.

EXAMPLES

Example 1

ADAM 12 immunoassay using MAbs A sandwich immunoassay based on two ADAM12-specific monoclonal antibodies was developed for the detection of human ^'ADAM12 in serum samples. One monoclonal antibody (clone 6E6) was coated on microtitration plates and the other (clone 8F8) was labelled with DELFIA Eu N1 chelate (PerkinElmer Cat n:o 1244-301) according to the manufacturer's instructions. Samples and calibrators were added in 25 μL to the plate followed by Eu- labelled monoclonal antibody in 100 μL (2μg/ml) of DELFIA Assay Buffer (PerkinElmer Cat n:o 1244-106) . After shaking for 2 hours at RT the plate was washed 4 times with DELFIA Wash solution (PerkinEimer Cat n:o 1244-114). Then DELFIA Enhancement solution (PerkinElmer Cat n:o 1244-104) was added (200 μL/well) and plate was shaken for 5 minutes and then measured in a time-resolved fluorometer. The results are shown in Figure 1.

The assay worked well but it was found that ADAM 12 concentrations in maternal serum samples decreased markedly upon storage. When serum samples were stored at +4°C for 2 days and 7 days there was a 10% and 30% decrease in ADAM12 concentrations, respectively. Results were even more troublesome when serum samples were stored at room temperature (21 "C). There was about 30-40% decrease and 60-70% decrease in ADAM12 concentrations after a storage of 2 days and 7 days, respectively.

Example 2 Labelling of Fab fragments with Eu chelate

Dimeric Fab fragments specific for human ADAM 12 obtained by screening against a commercially available library (AbD Serotec) were labelled with Europium chelates for use in ADAM12 assays. Labelling was performed at 4°C, pH 9.3, overnight. Fab52 StrepTag was labelled at 0.9 mg/mL using a 25- fold molar excess of DELFIA Eu-NI-ITC chelate having a reactive isothiocy- anato group (product number AD0001 , PerkinElmer). Just prior to use the Eu- N1-ITC chelate (20 mg, 32 μmoles) was dissolved in 1 mL water to give 32 mM Eu chelate and a calculated amount of the dissolved chelate was added to Fab solution. To adjust pH to 9.3, 1 M Na₂CU₃ pH 9 was added to a final con- centration of 0.1 M. If necessary pH was adjusted to 9.3 with 1 M NaOH. After overnight reaction at 4°C Eu-labelled Fab fragment was purified from free Eu chelate using a Superdex 200 column (GE Healthcare) and TSA buffer (50 mM Tris-HCI pH 7.8 containing 0.9% sodium chloride and 0.05% sodium azide. Purified labelled Fab fragment was pooled and then characterized by measur- ing Eu concentration and absorbance of the labelled Fab at 280 nm. Eu concentration was measured using DELFIA Enhancement solution. Next contribution of Eu chelate to absorbance at 280 nm could be subtracted and then Fab concentration could be calculated based on the molar absorptivity of 127 000. This protocol gave a labelling yield of 3.3 Eu chelate per Fab. Example 3

Epitope mapping of ADAM 12 FAb fragments

Epitopes of ADAM12 Fab fragments were mapped using synthetic peptide scanning. Spot synthesis of 20-mer peptides, overlapping by 15, was performed on cellulose membranes using Fmoc chemistry. Peptides covered the entire 739 amino acid sequence of human ADAM12 (SEQ ID NO: 1). After deprotection membranes were blocked with 0.1% casein in Tris-HCI buffered saline for 2 hours. Next membranes were incubated with biotinylated Fab fragments (10 μg/mL in the blocking buffer). After washing membranes were treated with horseradish peroxidase-conjugated streptavidin diluted in the blocking buffer. Following washes membranes were incubated in a luminol- based enhanced chemiluminescence detection mixture and exposed on X-ray film for various times (15 s to 2 min).

Fab fragments FAbI and FAb52 were found to bind to the following linear sequences in human ADAM12: Amino acids 445-475; Amino acids 560- 580; Amino acids 625-645; Amino acids 650-660: and Amino acids 360-380.

Amino acids 445-475 are in the disintegrin domain, amino acids 560-580, 625-645 and 650-660 are in the cysteine-rich domain, whereas amino acids 360-380 are located in the metalloprotease domain of human ADAM12.

Example 4

Sample stability studies with Fab fragments

Due to the poor stability of ADAM 12 in maternal serum samples in DELFIA ADAM 12 assay with Eu-labelled monoclonal antibody (clone 8F8), recombinant monoclonal dimeric Fab fragments (Fab1 and Fab52) were tested as tracers. Samples and calibrators were added in 25 μl_ to the plate followed by Eu-labelled Fab fragment in 100 μl_ (500ng/ml) of DELFIA Assay Buffer. After shaking for 2 hours at RT the plate was washed 4 times with DELFIA Wash solution. Following addition of DELFIA Enhancement Solution (200 μL/well) the plate was shaken for 5 minutes and then measured in a time- resolved fluorometer. Eu-labelled Fab fragments were found to be clearly superior to monoclonal antibody 8F8 concerning ADAM12 in maternal serum sample stability.

As shown in Figure 2 there is on average only 5-15% decrease in ADAM 12 concentration of serum samples after storage at 4°C and RT for up to 6 days. This improved sample stability allows the delivery of samples by post and courier. Additionally, samples stored at -20⁰C are stable when Fab fragments are used as tracers in DELFIA ADAM12 assay. This makes it possible to run retrospective studies or analyze samples collected during a period of 2-3 weeks in the first trimester of pregnancy and stored at -20⁰C.

Example 5

Homogeneous immunoassay for ADAM 12

Homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) assay for ADAM was developed. In TR-FRET assays a lantha- nide chelate with a long fluorescence lifetime (typically over 100 μs) and a conventional fluorescent label with a short lifetime (typically less than 5 ns) are used. A prerequisite for TR-FRET is that an excited lanthanide chelate is a donor giving energy to an acceptor, a conventional fluorescent label. Characteristic of TR-FRET is the fact that when energy transfer takes place, an acceptor gives fluorescence at its typical wavelength with a long lifetime (typically longer than 50 μs). Requirements for TR-FRET include that emission peak of a lanthanide chelate overlaps excitation peak of a fluorescent label and distance between a lanthanide chelate and an acceptor is no longer than 10-15 nm. For example, when one antibody labelled with a fluorescent Eu chelate and the other antibody labelled with a suitable acceptor bind to the same antigen, distance between the labels is short enough for an efficient energy transfer. On the other hand, unbound reagents in solution are not in close proximity and there is no energy transfer. Examples of suitable acceptors for a fluorescent Eu chelate include a fluorescent protein allophycocyanin (APC) and small- molecule fluorophores such as Hil_yte647, Alexa647 and Cy5. All these acceptors have an emission maximum at 650-680 nm.

Monoclonal antibody 6E6 was labelled with a fluorescent Eu chelate, LANCE Eu-WI 024-ITC chelate (PerkinEimer, catalog number AD0013) according to manufacturer's instructions. Labelling yield was 7 Eu-WI 024 che- lates per antibody. Fab51 StrepTag was labelled with HiLyte647 N- hydroxysuccinimide ester (Anaspec) according to manufacturer's instructions giving 3 HiLyte molecules per Fab fragment. Immunoassays were performed in microtitration plates with a total volume of 200 μL. Assay buffer was Tris-HCI based (50 mM, pH 7.8) containing 0.9% NaCI, 0.5% bovine serum albumin (BSA) and 0.05% bovine gamma-globulin. First samples, 50 μL sδrum or plasma, were added to wells followed by 150 μl_ assay buffer containing Eu- W1024-labelled monoclonal antibody 6E6 and HiLyte647-labelled Fab51. Final concentrations for Eu-labelled 6E6 and Hil_yte647-labelled Fab were 200 ng/mL and 400 ng/mL. After incubating for 1 hour at room temperature wells were measured in EnVision multilabel reader (PerkinElmer) using the factory- set LANCE Eu protocol (TR-FRET protocol for Eu chelate as a donor). Results at 665 nm reflect the amount of formed complex between the antigen, ADAM12, and both labelled reagents. Typically a sample having no ADAM12 gave 950 counts while samples containing ADAM 12 gave 3-20 000 counts de- pending on the concentration of ADAM12 in samples.

It will be obvious to a person skilled in the art that, as the technology advances, the inventive concept can be implemented in various ways. The invention and its embodiments are not limited to the examples described above but may vary within the scope of the claims.

Claims

1. An isolated epitope of human ADAM 12 comprising the amino acid sequence SEQ ID NO. 3 and at least one amino acid sequence selected from the group consisting of: SEQ ID NO. 4; SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.

2. A binding agent that specifically binds to an epitope of ADAM12 according to claim 1 , wherein the binding agent is an antibody or a fragment thereof.

3. The agent according to claim 2, wherein said fragment is a Fab fragment.

4. The agent according to claim 3, wherein said Fab fragment is dimeric.

5. A method for detecting ADAM12 in a biological sample, comprising: i) contacting said sample with a first binding agent capable of binding to

ADAM 12; ii) further contacting said sample with a second binding agent according to any one of claims 2-4, or a fragment thereof; iii) detecting the binding of ADAM12 to said first and second binding agents.

6. The method according to claim 5, wherein said first binding agent is a monoclonal antibody recognizing an epitope of ADAM12, which is different from the epitope according to claim 1.

7. The method according to claim 6, wherein said first binding agent is a monoclonal antibody, 6E6.

8. The method according to any one of claims 5-7, wherein said first binding agent is immobilized on a sold support and said second binding agent is labeled.

9. The method according to any one of claims 5-7, wherein said first binding agent is labeled, and said second binding agent is immobilized on a solid support.

10. A method for screening of conditions related to ADAM12, comprising: i) providing a sample obtained from a patient; ii) contacting said sample with an agent according to any one of claims 2-4, or a fragment thereof; iii) determining the level of ADAM12 in said sample; and iv) comparing the determined level of ADAM12 in said sample with normal levels of ADAM 12, wherein an abnormal level of ADAM 12 is indicative of said condition.

11. The method according to claim 10, wherein said condition is selected from the group consisting of fetal chromosomal anomaly, preeclampsia and cancer.

12. A kit for detecting ADAM 12, comprising a binding agent to any one of claims 2-4.

13. The kit according to claim 12, further comprising ADAM12 calibrators, assay buffers, reaction vials coated with an ADAM12 specific binding agent recognizing a second antigenic determinant of ADAM12.

14. The kit according to claim 13, wherein said binding agent is labeled for detection purposes.

15. The kit according to claim 14, wherein said binding agent is labeled by a Eu-label.