US20140148363A1

US20140148363A1 - Methods for Selecting Oocytes and Competent Embryos with High Potential for Pregnancy Outcome

Info

Publication number: US20140148363A1
Application number: US14/168,052
Authority: US
Inventors: Samir Hamamah; John De Vos; Said Assou
Original assignee: Institut National de la Sante et de la Recherche Medicale INSERM
Current assignee: Universite de Montpellier I; Institut National de la Sante et de la Recherche Medicale INSERM; Centre Hospitalier Universitaire de Montpellier CHUM
Priority date: 2009-04-17
Filing date: 2014-01-30
Publication date: 2014-05-29
Also published as: JP2012523826A; BRPI1015018A2; IL215662A0; EP3037554B1; ES2572830T3; AU2010237203A1; JP5828143B2; US20120077697A1; EP2419526B1; WO2010118991A1; EP2419526A1; ES2690453T3; CN102459635A; CA2758351A1; AU2010237203B2; US8754014B2; BRPI1015018B1; TR201815478T4; CA2758351C; BRPI1015018B8

Abstract

The present invention relates to a method for selecting a competent oocyte or a competent embryo.

Description

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

In assisted reproductive technology (ART), pregnancy and birth rates following in vitro fertilization (WF) attempts remain low. Indeed, 2 out of 3 IVF cycles fail to result in pregnancy (SART 2004) and more than 8 out of 10 transferred embryos fail to implant (Kovalevsky and Patrizio, 2005). In addition, more than 50% of IVF-born babies are from multiple gestations (Reddy et al., 2007). Preterm deliveries that result from multiple pregnancies caused by ART are estimated to account for approximately $890 million of U.S. health care costs annually (Bromer and Seli, 2008).
Subjective morphological parameters are still a primary criterion to select healthy embryos used for in IVF and ICST programs. However, such criteria do not truly predict the competence of an embryo. Many studies have shown that a combination of several different morphologic criteria leads to more accurate embryo selection (Balaban and Urman, 2006; La Sala et al., 2008; Scott et al., 2000). Morphological criteria for embryo selection are assessed on the day of transfer, and are principally based on early embryonic cleavage (25-27h post insemination), the number and size of blastomeres on day two or day three, fragmentation percentage and the presence of multi-nucleation in the 4 or 8 cell stage (Fenwick et al., 2002).
However, a recent study has shown that the selection of oocytes for insemination does not improve outcome of ART as compared to the transfer of all available embryos, irrespective of their quality (La Sala et al., 2008). There is a need to identify viable embryos with the highest implantation potential to increase IVF success rates, reduce the number of embryos for fresh replacement and lower multiple pregnancy rates.
For all these reasons, several biomarkers for embryo selection are currently being investigated (Haouzi et al., 2008; Pearson, 2006). As embryos that result in pregnancy differ in their metabolomic profiles compared to embryos that do not, some studies are trying to identify a molecular signature that can be detected by non-invasive evaluation of the embryo culture medium (Brison et al., 2004; Gardner et al., 2001; Sakkas and Gardner, 2005; Seli et al., 2007; Zhu et al., 2007).
Genomics are. also providing vital knowledge of genetic and cellular function during embryonic development. (McKenzie et al., 2004) and (Feuerstein et al., 2007) have reported, that the expression of several genes in cumulus cells, such as cyclooxygenase 2 (COX2), was indicative of oocyte and embryo quality. Gremlin 1 (GREM 1), hyaluronic acid synthase 2 (HAS2), steroidogenic acute regulatory protein (STAR), stearoyl-coenzyme A desaturase 1 and 5 (SCDI and 5), amphiregulin (AREG) and pentraxin 3 (PTX3) have also been shown to be positively correlated with embryo quality (Zhang et al. 2005). More recently, the expression of glutathione peroxidase 3 (GPX3), chemokine receptor 4 (CXCR4), cyclin D2 (CCND2) and catenin delta I (CTNND1) in human cumulus cells have been shown to be inversely correlated with embryo quality, based on early-cleavage rates during embryonic development (van Montfoort et al. 2008). But, despite the fact that early cleavage has been shown to be a reliable biomarker for predicting pregnancy (Lundin et al., 2001; Van Montfoort et al., 2004; Yang et al., 2007), gene expression profiles of cumulus cells had not been studied with respect to pregnancy outcome.

SUMMARY OF THE INVENTION

The present invention relates to a method for selecting a competent oocyte, comprising a step of measuring the expression level of 45 genes in a cumulus cell surrounding said oocyte, wherein said genes are WNT6, LRCH4, PAX8, CABP4, PDE5A, BCL2L11, PCK 1, TCF20, SLAMF6, EPOR, CACNG6, NLRP1, PECAM1, NOS1, ATF3, KRTAP8, GRIK5, SLC24A3, SLC5A12, SLCA10A2, SLCO1A2, SLC25A5, MG29, NLGN2. PRKACA, FOSB, SIAT6, LOXL2, PRFI, ADPRH. APBB3, EGR3, CNR2, IFITM1, PLA2G5, CAMTA1, SOX4, NFIB, NFIC, RBMS1, G0S2, FAT3, SLC40A1, GPC6 and IGFIR.
The present invention also relates to a method for selecting a competent embryo, comprising a step of measuring the expression level of 45 genes in a cumulus cell surrounding the embryo, wherein said genes are WNT6. LRCH4, PAX8, CABP4, PDE5A, BCL2L11, PCK1, TCF20, SLAMF6, EPOR, CACNG6, NLRP1, PECAM1, NOS1, ATF3, KRTAP8, GRIK5, SLC24A3, SLC5A12, SLCA10A2, SLCO1A2. SLC25A5, MG29, NLGN2, PRKACA, FOSB, SIAT6, LOXL2, PRF1, ADPRH, APBB3, EGR3, CNR2, IFITM1, PLA2G5, CAMTA1, SOX4, NFIB, NFIC, RBMS1, GOS2, FATS, SLC40A1, GPC6 and IGFIR.
The present invention also relates to a method for selecting a competent oocyte or a competent embryo, comprising a step of measuring in a cumulus cell surrounding said oocyte or said embryo the expression level of one or more genes selected from the groups A, B or C. wherein group A consists of PCK1, ADPRH, CABP4, SLAMF6, CAMTA1, CSPG2, and PRF1; group B consists of FOSB, NLGN2, PDE5A, PLA2G5, GPC6, and EGR3; and group C consists of NFIB, NFIC, IGFIR, G0S2, GIRK5 and RBMS1.
Overexpression of one or more genes selected from group A is predictive of a competent oocyte or embryo leading to pregnancy. Overexpression of one or more genes selected from group B is predictive of a non competent oocyte or embryo, the embryo being unable to implant. Overexpression of one or more genes selected from group C is predictive of a non competent oocyte or embryo due to early embryo arrest.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have determined as set of genes expressed in cumulus cells that are biomarkers for embryo potential and pregnancy outcome. They demonstrated that genes expression profile of cumulus cells which surrounds oocyte correlated to different pregnancy outcomes, allowing the identification of a specific expression signature of embryos developing toward pregnancy. Their results indicate that analysis of cumulus cells surrounding the oocyte is a non-invasive approach for embryo selection.

Set of Predictive Genes

All the genes pertaining to the invention are known per se, and are listed in the below Tables A and B. Tables A and B present the set of genes whose combined expression profile has been shown to be informative for selecting a competent oocyte or for selecting a competent embryo with a high implantation potential leading to pregnancy.

TABLE A

set of predictive genes.

Gene		Gene
Symbol	Gene name	ID

WNT6	wingless-type MMTV integration site family, member 6	7475
LRCH4	leucine-rich repeats and calponin homology (CH) domain containing	4034
PAX8	paired box 8	7849
CABP4	calcium binding protein 4	57010
PDE5A	phosphodiesterase 5A, cGMP-specific	8654
BCL2L11	BCL2-like 11 (apoptosis facilitator)	10018
PCK1	phosphoenolpyruvate carboxykinase 1 (soluble)	5105
TCF20	transcription factor 20 (AR1)	6942
SLAMF6	SLAM family member 6	114836
EPOR	erythropoietin receptor	2057
CACNG6	calcium channel, voltage-dependent, gamma subunit 6	59285
NLRP1	NLR family. pyrin domain containing 1	22861
PECAM1	platelet/endothelial cell adhesion molecule	5175
NOS1	nitric oxide synthase 1 (neuronal)	4842
ATF3	activating transcription factor 3	467
KRTAP8	keratin associated protein 8-1	337879
GRIK5	glutamate receptor, ionotropic, kainate 5	2901
SLC24A3	solute carrier family 24 (sodium/potassium/calcium exchanger), member 3	57419
SLC5A12	solute carrier family 5 (sodium/glucose cotransporter). member 12	159963
SLCA10A2	Solute carrier family 10 (sodium/bile acid cotransporter family), member 2	6555
SLCO1A2	solute carrier organic anion transporter family, member 1A2	6579
SLC25A5	solute carrier family 25 (mitochondrial carrier; adenine nucleotide	292
	translocator), member 5
MG29 or	synaptophysin-like 2	284612
SYPL2
NLGN2	neuroligin 2	57555
PRKACA	protein kinase, cAMP-dependent, catalytic, alpha	5566
FOSB	FBJ murine osteosarcoma viral oncogene homolog B	2354
SIAT6	ST3 beta-galactoside alpha-2,3-sialyltransferase 3	6487
LOXL2	lysyl oxidase-like 2	4017
PRF1	perforin 1 (pore forming protein)	5551
ADPRH	ADP-ribosylarginine hydrolase	141
APBB3	amyloid beta (A4) precursor protein-binding, family B, member 3	10307
EGR3	early growth response 3	1960
CNR2	cannabinoid receptor 2 (macrophage)	1269
IFITM1	interferon induced transmembrane protein 1 (9-27)	8519
PLA2G5	phospholipase A2, group V	5322
CAMTA1	calmodulin binding transcription activator 1	23261
SOX4	SRY (sex determining region Y)-box 4	6659
NFIB	nuclear factor I/B	4781
NFIC	nuclear factor I/C (CCAAT-binding transcription factor)	4782
RBMS1	RNA binding motif, single stranded interacting protein 1	5937
G0S2	G0/G1switch 2	50486
FAT3	FAT tumor suppressor homolog 3 (Drosophila)	120114
SLC40A1	solute carrier family 40 (iron-regulated transporter), member 1	30061
GPC6	glypican 6	10082
IGF1R	insulin-like growth factor 1 receptor	3480

An object of the invention relates to a method for selecting a competent oocyte, comprising a step of measuring the expression level of 45 genes in a cumulus cell surrounding said oocyte, wherein said genes are WNT6, LRCH4, PAX8, CABP4, PDE5A, BCL2L11, PCK1, TCF20, SLAMF6, EPOR, CACNG6, NLRP1, PECAMI, NOS1, ATF3, KRTAP8, GRIK5, SLC24A3, SLC5A12, SLCA10A2, SLCO1A2, SLC25A5, MG29, NLGN2, PRKACA, FOSB, SIAT6, LOXL2, PRF1, ADPRH, APBB3, EGR3, CNR2, IFITM1, PLA2G5, CAMTA1, SOX4, NFIB, NFIC, RBMS1, G0S2, FAT3, SLC40A1, GPC6 and IGFIR.
A used herein the term “competent oocyte” refers to a female gamete or egg that when fertilized produces a viable embryo with a high implantation rate leading to pregnancy.
According to the invention, the oocyte may result from a natural cycle, a modified natural cycle or a stimulated cycle for cIVF or ICSI. The term “natural cycle” refers to the natural cycle by which the female or woman produces an oocyte. The term “modified natural cycle” refers to the process by which, the female or woman produces an oocyte or two under a mild ovarian stimulation with GnRH antagonists associated with recombinant FSH or hMG. The term “stimulated cycle” refers to the process by which a female or a woman produces one ore more oocytes under stimulation with GnRH agonists or antagonists associated withrecombinant FSH or hMG.
The term “cumulus cell” refers to a cell comprised in a mass of cells that surrounds an oocyte. These cells are believed to be involved in providing an oocyte some of its nutritional, energy and or other requirements that are necessary to yield a viable embryo upon fertilization.
The methods of the invention may further comprise a step consisting of comparing the expression level of the genes in the sample with a control, wherein detecting differential in the expression level of the genes between the sample and the control is indicative whether the oocyte is competent. The control may consist in sample comprising cumulus cells associated with a competent oocyte or in a sample comprising cumulus cells associated with an unfertilized oocyte.
The methods of the invention are applicable preferably to women but may be applicable to other mammals (e.g., primates, dogs, cats, pigs, cows . . . ).
The methods of the invention are particularly suitable for assessing the efficacy of an in vitro fertilization treatment. Accordingly the invention also relates to a method for assessing the efficacy of a controlled ovarian hyperstimulation (COS) protocol in a female subject comprising:
i) providing from said female subject at least one oocyte with its cumulus cells;
ii) determining by a method of the invention whether said oocyte is a competent oocyte.
Then after such a method, the embryologist may select the competent oocytes and in vitro fertilized them through a classical in vitro fertilization (cIVF) protocol or under an intracytoplasmic sperm injection (ICSI) protocol.
A further object of the invention relates to a method for monitoring the efficacy of a controlled ovarian hyperstimulation (COS) protocol comprising:
i) isolating from said woman at least one oocyte with its cumulus cells under natural, modified or stimulated cycles;
ii) determining by a method of the invention whether said oocyte is a competent oocyte;
iii) and monitoring the efficacy of COS treatment based on whether it results in a competent oocyte.
The COS treatment may be based on at least one active ingredient selected from the group consisting of GnRH agonists or antagonists associated with recombinant FSH or hMG.
The present invention also relates to a method for selecting a competent embryo, comprising a step of measuring the expression level of 45 genes in a cumulus cell surrounding the embryo, wherein said genes are WNT6, LRCH4, PAX8, CABP4, PDE5A, BCL2L11, PCK1, TCF20, SLAMF6, EPOR, CACNG6, NLRP1, PECAM1, NOS1, ATF3, KRTAP8, GRIK5, SLC24A3, SLC5A12. SLCA10A2, SLCO1A2, SLC25A5, MG29, NLGN2, PRKACA, FOSB, SIAT6, LOXL2, PRFI, ADPRH. APBB3. EGR3, CNR2, IFITM1, PLA2G5, CAMTA1, SOX4, NF1B, NF1C, RBMS1, G0S2, FAT3, SLC40A1, GPC6 and IGFIR.
The term “embryo” refers to a fertilized oocyte or zygote. Said fertilization may intervene under a classical in vitro fertilization (cIVF) or under an intracytoplasmic sperm injection (ICSI) protocol.
The term “classical in vitro fertilization” or “cIVF” refers to a process by which oocytes are fertilised by sperm outside of the body, in vitro. IVF is a major treatment in infertility when in vivo conception has failed. The term “intracytoplasmic sperm injection” or “ICSI” refers to an in vitro fertilization procedure in which a single sperm is injected directly into an oocyte. This procedure is most commonly used to overcome male infertility factors, although it may also be used where oocytes cannot easily be penetrated by sperm, and occasionally as a method of in vitro fertilization, especially that associated with sperm donation.
The term “competent embryo” refers to an embryo with a high implantation rate leading to pregnancy. The term “high implantation rate” means the potential of the embryo when transferred in uterus, to be implanted in the uterine environment and to give rise to a viable foetus, which in turn develops into a viable offspring absent a procedure or event that terminates said pregnancy.
The methods of the invention may further comprise a step consisting of comparing the expression level of the genes in the sample with a control, wherein detecting differential in the expression level of the genes between the sample and the control is indicative whether the embryo is competent. The control may consist in sample comprising cumulus cells associated with an embryo that gives rise to a viable foetus or in a sample comprising cumulus cells associated with an embryo that does not give rise to a viable foetus.
It is to note that the methods of the invention leads to an independence from morphological considerations of the embryo. Two embryos may have the same morphological aspects but by a method of the invention may present a different implantation rate leading to pregnancy.
The methods of the invention are applicable preferably to women but may he applicable to other mammals (e.g. primates, dogs, cats, pigs, cows . . . ).
The present invention also relates to a method for determining whether an embryo is a competent embryo, comprising a step consisting in measuring the expression level of 45 genes in a cumulus cell surrounding the embryo, wherein said genes are WNT6, LRCH4, PAX8, CABP4, PDE5A, BCL2L11, PCK1, TCF20, SLAMF6, EPOR, CACNG6, NLRP1, PECAM1, NOS1, ATF3, KRTAP8, GRIK5, SLC24A3, SLC5A12, SLCA10A2, SLCO1A2, SLC25A5, MG29, NLGN2, PRKACA, FOSB, SIAT6, LOXL2, PRFI, ADPRH, APBB3, EGR3, CNR2. IFITM1, PLA2G5, CAMTAI, SOX4, NFIB, NFIC, RBMS1, G0S2, FAT3, SLC40A1, GPC6 and IGFIR.
The present invention also relates to a method for determining whether an embryo is a competent embryo, comprising:
i) providing an oocyte with its cumulus cells
ii) in vitro fertilizing said oocyte
iii) determining whether the embryo that results from step ii) is competent by determining by a method of the invention whether said oocyte of step i), is a competent oocyte.
The present invention also relates to a method for selecting a competent oocyte or a competent embryo, comprising a step of measuring in a cumulus cell surrounding said oocyte or said embryo the expression level of one or more genes selected from the groups A, B or C, wherein group A consists of PCK1, ADPRH, CABP4, SLAMF6, CAMTAI, CSPG2. and PRFI; group B consists of FOSB, NLGN2, PDE5A, PLA2G5, GPC6, and EGR3; and group C consists of NFIB. NFIC, IGFIR, G0S2, GIRK5 and RBMS 1.
Overexpression of one or more genes selected from group A is predictive of a competent oocyte or embryo leading to pregnancy. Overexpression of one or more genes selected from group B is predictive of a non competent oocyte or embryo, the embryo being unable to implant. Overexpression of one or more genes selected from group C is predictive of a non competent oocyte or embryo due to early embryo arrest. Said one or more genes may be selected for example from group A alone, group B alone or group C alone. Typically, 1, 2, 3, 4, 5, 6 or 7 genes may be selected from group A. Typically, 1, 2, 3, 4, 5, or 6 genes may be selected from group B. Typically, 1, 2, 3, 4, 5, or 6 genes may be selected from group C. Alternatively, said genes may be selected for example from groups A and B, from groups A and C, from groups B and C, or from groups A, B and C. Typically, 1, 2, 3, 4, 5, 6 or 7 genes may be selected from group A, and 0, 1, 2, 3, 4, 5, or 6 genes may be selected from group B and 0, 1, 2, 3, 4, 5, or 6 genes may be selected from group C. Typically, 0, 1, 2, 3, 4, 5, 6 or 7 genes may be selected from group A, and 1, 2, 3, 4, 5, or 6 genes may be selected from group B and 0, 1, 2, 3, 4. 5, or 6 genes may be selected from group C. Typically, 0, 1, 2, 3, 4, 5, 6 or 7 genes may be selected from group A, and 0, 1, 2, 3, 4, 5, or 6 genes may be selected from group B and 1, 2, 3, 4, 5, or 6 genes may be selected from group C.
The methods of the invention are particularly suitable for enhancing the pregnancy outcome of a female. Accordingly the invention also relates to a method for enhancing the pregnancy outcome of a female comprising:
i) selecting a competent embryo by performing a method of the invention
iii) implanting the embryo selected at step i) in the uterus of said female.
The method as above described will thus help embryologist to avoid the transfer in uterus of embryos with a poor potential for pregnancy out come.
The method as above described is also particularly suitable for avoiding multiple pregnancies by selecting the competent embryo able to lead to an implantation and a pregnancy.
In all above cases, the methods described the relationship between genes expression profile of cumulus cells and embryo and pregnancy outcomes.
Methods for Determining the Expression Level of the Genes of the Invention:
Determination of the expression level of the genes as above described in Tables A and B can be performed by a variety of techniques. Generally, the expression level as determined is a relative expression level.
More preferably, the determination comprises contacting the sample with selective reagents such as probes, primers or ligands, and thereby detecting the presence, or measuring the amount, of polypeptide or nucleic acids of interest originally in the sample. Contacting may be performed in any suitable device, such as a plate, microtiter dish, test tube, well, glass, column. and so forth. In specific embodiments, the contacting is performed on a substrate coated with the reagent, such as a nucleic acid array or a specific ligand array. The substrate may be a solid or semi-solid substrate such as any suitable support comprising glass, plastic, nylon, paper, metal, polymers and the like. The substrate may be of various forms and sizes, such as a slide, a membrane, a bead, a column, a gel, etc. The contacting may be made under any condition suitable for a detectable complex, such as a nucleic acid hybrid or an antibody-antigen complex, to be formed between the reagent and the nucleic acids or polypeptides of the sample.
In a preferred embodiment, the expression level may be determined by determining the quantity of mRNA.
Methods for determining the quantity of mRNA are well known in the art. For example the nucleic acid contained in the samples (e.g., cell or tissue prepared from the patient) is first extracted according to standard methods. for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis) and/or amplification (e.g., RT-PCR). Preferably quantitative or semi-quantitative RT-PCR is preferred. Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous.
Other methods of Amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).
Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin).
Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. Primers typically are shorter single-stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are “specific” to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50% formamide, 5×or 6×SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
The nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes the components necessary to determine if amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.
In a particular embodiment, the methods of the invention comprise the steps of providing total RNAs extracted from cumulus cells and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semi-quantitative RT-PCR.
In another preferred embodiment, the expression level is determined by DNA chip analysis. Such DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs. To determine the expression level, a sample from a test subject, optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microanarray surface. The labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210)
In this context, the invention further provides a DNA chip comprising a solid support which carries nucleic acids that are specific to the genes listed in table A or B.
Other methods for determining the expression level of said genes include the determination of the quantity of proteins encoded by said genes.
Such methods comprise contacting the sample with a binding partner capable of selectively interacting with a marker protein present in the sample. The binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.
The presence of the protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, etc. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
The aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with an antibody against the protein to be tested. A biological sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a delectably labeled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.
Alternatively an immunohistochemistry (IHC) method may be preferred. IHC specifically provides a method of detecting targets in a sample or tissue specimen in situ. The overall cellular integrity of the sample is maintained in IHC, thus allowing detection of both the presence and location of the targets of interest. Typically a sample is fixed with formalin, embedded in paraffin and cut into sections for staining and subsequent inspection by light microscopy. Current methods of IHC use either direct labeling or secondary antibody-based or hapten-based labeling. Examples of known IHC systems include, for example, EnVision™ (DakoCytomation). Powervision(R) (Immunovision. Springdale. Ariz.), the NBA™ kit (Zymed Laboratories Inc., South San Francisco, Calif.). HistoFine(R) (Nichirei Corp, Tokyo, Japan).
In particular embodiment, a tissue section (e.g. a sample comprising cumulus cells) may be mounted on a slide or other support after incubation with antibodies directed against the proteins encoded by the genes of interest. Then, microscopic inspections in the sample mounted on a suitable solid support may be performed. For the production of photomicrographs, sections comprising samples may be mounted on a glass slide or other planar support, to highlight by selective staining the presence of the proteins of interest.
Therefore IHC samples may include, for instance: (a) preparations comprising cumulus cells (b) fixed and embedded said cells and (c) detecting the proteins of interest in said cells samples. In some embodiments, an IHC staining procedure may comprise steps such as: cutting and trimming tissue, fixation, dehydration, paraffin infiltration, cutting in thin sections, mounting onto glass slides, baking, deparaffination, rehydration, antigen retrieval, blocking steps, applying primary antibodies, washing, applying secondary antibodies (optionally coupled to a suitable detectable label), washing, counter staining, and microscopic examination.
The invention also relates to a kit for performing the methods as above described, wherein said kit comprises means for measuring the expression level the levels of the genes of Tables A or B that are indicative whether the oocyte or the embryo is competent.
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

EXAMPLES

A NON-INVASIVE TEST FOR ASSESSING EMBRYO POTENTIAL BY GENE EXPRESSION PROFILES OF HUMAN CUMULUS CELLS

Example 1

Material & Methods:
Patients and IVF treatment: In this retrospective study, normo-responder patients (n=30) aged of 30.9 years ±2.5 and referred to our centre for ICSI (Intra Cytoplasmic Sperm Injection) for male infertility factor were studied. Patients were stimulated with a combination of GnRH agonist or antagonist with recombinant FSH (GonalF, Puregon; respectively of Merck-Serono and Organon) or with hMG (Menopur, Ferring). Ovarian response was evaluated by serum estradiol level and ultrasound examination to monitor follicle development. Retrieval of oocytes was performed 36 hours after hCG administration (5000 IU), under ultrasound guidance.
Assessment of embryo quality: On day 2 and 3 postmicroinjection, the quality parameters of individually cultured embryo were evaluated using the number of blastomeres and the degree of fragmentation as criteria (grade 1-2: equally sized blastomeres and 0-20% fragmentation, grade 3-4: no equally sized blastomeres and more than 20% fragmentation. A top-quality embryo was defined on day 3 as 6-8 cells, equally sized blastomeres and no fragmentation. One or two embryos were transferred on day 3 after oocyte retrieval. Clinical pregnancy was evaluated two and six weeks after embryo transfer based respectively on serum Beta-hCG and ultrasound examination (presence of gestational sac with heart beat).
Cumulus cells: All cumulus cells (CC) samples were frozen on egg collection day. Then, one to 3 CC samples per patient were randomly selected for microarray analysis. A total of 50 CC samples were collected from 50 single oocytes and analyzed individually: 34 CC from grade 1-2 embryos (n=20 patients), 11 CC from grade 3-4 embryos (n=10 patients) and 5 CC from unfertilized oocytes (n=5 patients) (Table 1).

TABLE 1

The Characteristics of cumulus cells samples in this study

	30 patients	5 Patients
	45 CC	5 CC

G1/2 (34 CC)

G3/4 (11 CC)

cumulus cells from

	P+	P−	NT	unfertilized oocyte (5 CC)

chips nbr	18	16	11	5
patients nbr	11	9	10	5
CC nbr	18	16	11	5

CC: cumulus cells,
P+: cumulus cells from embryos with positive pregnancy outcome,
P−: cumulus cells from embryos without pregnancy outcome,
G1/2: cumulus cells from grade 1-2 embryos,
G3/4: cumulus cells from grade 3-4 embryos,
NT: no transfer.

The data analysis was performed under double blind conditions in which pregnancy outcome was disclosed only after microarrays were hybridized. Regarding pregnancy outcome, the 45 CC from fertilized oocytes included 16 CC from grade 1-2 embryos that did not result into pregnancy (n=9 patients), 18 CC associated with a positive pregnancy outcome (n=11 patients) and 11 CC from grade 3-4 embryos that were not transferred. Cumulus cells were stripped immediately following oocyte recovery (<40 h post hCG administration). Cumulus cells were mechanically removed and washed in culture medium and immediately frozen at −80° C. in RLT RNA extraction buffer (RNeasy kit, Qiagen, Valencia, Calif., USA) before RNA extraction.
Granulosa cells: An independent group of normo responder patients (n=8) (age 34.8 years ±3.2) referred for ICSI program for male infertility factor was selected for granulosa cells collection (8 samples). Immediately after oocyte recovery, follicular fluids from matures follicles (>17 mm) of the same patient were pooled, after removal of the cumulus oocyte complex and diluted in ⅓ volume of HBSS solution (BioWhittaker) in 50 ml batches, representing one sample. Granulosa cells purification was adapted from the protocol by (Kolena et al., 1983). Following a 20 min. centrifugation at 500 g in swinging buckets, granulosa cells were collected on a Ficoll cushion (12 ml Lymphocyte separation medium, BioWhittaker). They were successively washed in HBSS and PBS, incubated 5 min. in blood lysis buffer (KHCO₃10 mM, NH₄Cl 150 mM, EDTA 0.1 mM) to remove red blood cells, counted and pelleted in PBS before lysis in RLT buffer (Quiagen) and storage at −80° C. The number of follicular puncture and the number of purified granulosa cells ranged from 6 to 12 and from 2 10⁶to 9 10⁶respectively.
Complementary RNA (cRNA) preparation and microarray hybridization: CC and granulosa cells RNA was extracted using the micro RNeasy Kit (Qiagen). The total RNA quantity was measured with a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies Inc., Delaware, USA) and RNA integrity was assessed with an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, Calif., USA). cRNA was prepared with two rounds of amplification according to the manufacturer's protocol “double amplification” (Two-Cycle cDNA Synthesis Kit, Invitrogen) starting from total RNA (ranging from 70 ng to 100 ng). cRNA obtained after the first amplification ranged from 0.1 μg/μl to 1.9 μg/μl and after the second amplification ranged from 1.6 μg/μl to 4.5 μg/μl.
Labelled fragmented cRNA (12 μg) was hybridized to oligonucleotide probes on an Affymetrix HG-U133 Plus 2.0 array containing 54 675 sets of oligonucleotide probes (“probeset”) which correspond to =30 000 unique human genes or predicted genes. Each cumulus and granulosa sample was put individually on a microarray chip.
Data processing: Scanned GeneChip images were processed using Affymetrix GCOS 1.4 software to obtain an intensity value and a detection call (present, marginal or absent) for each probeset, using the default analysis settings and global scaling as first normalization method, with a trimmed mean target intensity value (TGT) of each array arbitrarily set to 100. Probe intensities were derived using the MAS5.0 algorithm. This algorithm also determines whether a gene is expressed with a defined confidence level or not (“detection call”). This “call” can either be “present” (when the perfect match probes are significantly more hybridized than the mismatch probes, p-value <0.04), “marginal” (for p-values >0.04 and <0.06) or “absent” (p-value >0.06). The microarray data were obtained in our laboratory in agreement with the Minimal Information about a Microarray Experiment MIAME recommendations (Brazma et al. 2001).
Data analysis and visualisation: Significant Analysis of microarrays (SAM) (Tusher et al., 2001) (http://www-stat.stanford.edu/-tibs/SAM/) was used to identify genes whose expression varied significantly between sample groups. SAM provides mean or median fold change values (FC) and a false discovery rate (FDR) confidence percentage based on data permutation (mean fold change >2 and FDR <5%). Array analysis allowing the comparison of gene expression profile. between cumulus cell samples and granulosa cell samples is first based on the significant RNA detection (detection call “present” or “absent”) and then, submitted to a SAM (Significant Analysis of microarrays) to identify genes whose expression varied significantly between sample groups. To perform the comparison of gene expression profile between cumulus cell samples according embryonic quality and/or pregnancy outcome. a non-supervised selection of probesets using a variation coefficient (CV ≧40%) and a Absent/Present “detection call” filter was performed before the SAM. To compare profile expression of cumulus cells from altered (grade 3-4) and good (grade 1-2) embryonic development, or from embryos leading, or not, to a pregnancy, we performed an unsupervised classification with both principal component analysis (PCA) and hierarchical clustering (de Hoon et al., 2004; Eisen et al., 1998). The PCA involved original scripts based on the R statistics software through the RAGE web interface (http://rage.montp.inserm.fr) (Reme et al., 2008). Hierarchical clustering analysis based on the expression levels of varying probes were performed with the CLUSTER and TREEVIEW software packages. To uncover functional biological networks and top canonical pathways, we imported gene expression signatures into the Ingenuity Pathway's Analysis (IPA) Software (Ingenuity Systems, Redwood City, Calif., USA).
Quantitative RT-PCR analyses: For qRT-PCR analysis, 10 CC samples used in the microarray experiments were selected according to their pregnancy outcome (5 CC samples associated to a negative outcome and 5 to a positive outcome corresponding to 10 patients). Labelled cRNA (1 μg) from the patient was used to generate first strand cDNA. These cDNAs (5 μl of a 1/10 dilution) were used for real-time quantitative PCR reactions according to the manufacturer's recommendations (Applied Biosytems). The 20 μl reaction mixture consisted of cDNA (5 μl), 1 μM of primers and 10 μl of Taqman Universal PCR Master Mix (Applied Biosystem). The amplification was measured during 40 cycles with an annealing temperature at 60° C. The amount of PCR product produced in every cycle step of the PCR reaction is monitored by TaqMan probe. A threshold is set in the exponential phase of the amplification curve, from which the cycle number (“Ct” for “Cycle Threshold”) is read off. The Ct-value is used in the calculation of relative mRNA transcript levels. Effectiveness (E) of the PCR was measured. This effectiveness is obtained by a standard curve corresponding to the primers used. Quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) was performed using the ABI Prism 7000 sequence detection system (Applied Biosystems) and normalized to PGKI for each sample using the following formula: E_{tested primer} ^ΔCt/E_PGKI ^ΔCt(E=10^−1/slope), ΔCt=Ct control−Ct unknown, control=one CC sample of the non-pregnant group). Each sample was analysed in duplicate, and multiple water blanks were included with the analysis.
Results
Gene expression profile of CC according to embryo outcome: To identify a gene expression profile in CC that correlated with embryo outcome, we established a gene expression signature for each outcome category: CC of unfertilized oocytes, CC from oocytes that resulted in embryo development but extensive fragmentation (grade 3-4), and CC from oocytes that resulted in embryo development with no or limited fragmentation (grade 1-2). Granulosa cells samples were taken as a reference tissue (control). Indeed, granulosa cells are cells closely related to CC as opposed to other adult tissues. The use of this reference tissue lowered the number of differentially expressed genes related to crude lineage differences, thus facilitating the identification of subtle variation in the CC/oocyte interplay. A SAM analysis showed that 2605 genes were upregulated in the unfertilized group, 2739 in the grade ¾ group and 2482 in the grade 1-2 group with a FDR <5% . Conversely, 4270, 4349 and 4483 genes, were downregulated, respectively. These lists of genes were then intersected to determine their overlap. While 449 up and 890 down expressed genes were in common in all three groups, each category displayed a specific gene expression profile. Interestingly, 860 up-regulated genes, including for example Galanin and Gap Junction AS (GJA5). and 1416 down-regulated genes, including HLA-G and EGRI were specifically modulated in cumulus cells associated to a good morphological embryonic quality. It must be noted that although the grade 1-2 group displayed a strong gene expression profile, this group was heterogeneous regarding to pregnancy outcome and included 18 CC samples associated with embryos that resulted in pregnancy (including 4 twin pregnancies) but also 16 CC samples associated with embryos that failed to give rise to pregnancy.
Gene expression profile of CC according to pregnancy outcome: CC samples were therefore compared according to the pregnancy outcome. A SAM analysis delineated a “pregnancy outcome” list of 630 genes that varied significantly (FDR <5%) between the two group of patients (pregnancy versus no pregnancy). PCA and hierarchical clustering confirmed that this 630 gene list indeed segregated a majority of CC samples associated with pregnancy from from those associated with no pregnancy. Of note, genes from the “pregnancy outcome” list were predominantly upregulated in samples associated with a good outcome. The “pregnancy outcome” expression signature was particularly marked in a sub-group of 10 CC samples from embryos associated to the “pregnancy” group.
Functional annotation of the Pregnancy Outcome gene list: To investigate biological processes correlated to embryo achieving pregnancy. Ingenuity and Pubmed databases were used to annotate the 630 genes from the “pregnancy outcome” gene list. Among genes whose overexpression is associated with pregnancy, the most significantly overrepresented pathways were “oxidative stress”, “TR/RXR activation”, “G2/M transition of the cell cycle”, “xenobiotic metabolism” and “NFKappaB” signalling. Among these pathways, the most representative genes were interleukins, chemokines, adptator proteins and kinases: IL1Beta (×4.5 in pregnancy samples versus no pregnancy, P=0.001), IL16 (×4.8, P=0.001), IL8 (×2.6, P=0.007), ILIRN (×2.1, P=0.0051), IL17RC (×3.6, P=0.001), TIRAP (×8.0, P=0.001), CXCL12 (×3.1, P=0.001), CCR5 (×2.6, P=0.0051), and PCK1 (×3.4, P=0.001). Strikingly, numerous genes involved in the regulation of apoptosis were significantly modulated in CC samples from oocytes resulting in a pregnancy. These genes were BCL2L11 (×6.9, P<0.001), CRADD (×2, P=0.0036), NEMO (×4.6, P<0.001), BCL10 (×3.1, P=<0.001), SERPINB8 (×9.1, P<0.001). and TNFSF13 (×2.5, P=0.0038),
On the other hand, genes associated with no pregnancy were correlated with the following pathways: G2/M DNA damage and checkpoint regulation of the cell cycle, “Sonic hedgehog”, “IGF-1”, “complement system” and “Wnt/Beta-catenin” Representative genes correlated with no pregnancy included NFIB (×0.3, P<0.001), MAD2L1 (×0.4, P<0.001) and IGFIR (×0.4, P<0.001).
Candidate genes expressed in CC for embryo potential: The SAM analysis of CC according to pregnancy outcome identified the 45 genes of Table A that are biomarkers for embryo potential that would differentiate between oocytes that produced embryos resulting in a pregnancy versus those that did not result in pregnancy based on genes expression of CC analysis. QRT-PCR was used to confirm independently the microarray data. We analyzed the differential expression of 36 up-regulated genes and 9 down-regulated genes between CC from grade 1-2 embryos did not achieve pregnancy and CC from grade 1-2 embryos achieving pregnancy.
Conclusion:
In most mammalian species including human, the cumulus cells which surrounds the oocyte are still present at the time of fertilization in the oviduct and remain until embryonic implantation. Extracellular matrix remodelling within and around the cumulus probably plays a key role in both of these steps. In this respect, we identified 45 genes expressed in cumulus cells that are biomarkers for embryo potential and pregnancy outcome. In our study, we demonstrated that genes expression profile of CC which surrounds oocyte correlated to different outcomes, allowing the identification of a specific expression signature of embryos developing toward pregnancy. In conclusion, we found a differential gene expression between human cumulus cells from oocytes resulting in different pregnancy outcome from patients referred for ICSI or IVF. Our results indicate that analysis of cumulus cells surrounding the oocyte is a non-invasive approach for embryo selection. Typically CC can be collected immediately after oocyte pick-up, the CC can be analyzed with a genomic test (G-test) to assess the potential of the embryo, and the embryo can be then be selected for fresh replacement based on the G-test results.

Example 2

In order to test the reliability of the 45 gene list, we conducted a prospective study including young (<36 years) normal responder patients referred to our centre for ICSI for male infertility. The embryo selection occurred either according to the gene expression profile in CCs (group 1) or to morphological aspects (group 2 used as control). For each group, two embryos were replaced. For the first 60 patients (30 patients/group), on egg collection day, in group I, each CC sample was collected individually and processed for gene expression analysis. CC samples (n=267) were analyzed. Quantitative RT-PCR analysis was performed to measure the relative abundance of the transcripts of interest genes in CCs, and expression data for all biomarkers were obtained from all samples. All patients in both groups had a fresh embryo transfer on day 3. The comparison between the 2 groups reveals significant differences for implantation and ongoing pregnancy rates/pick up (40.0% vs. 26.7% and 70.0% vs. 46.7; p<0.05, respectively). We noted 5 twin pregnancies in group I versus 0 in the group 2 used as control. In addition, we observed that there was no relationship between morphological aspects and the CC gene expression profile. On the basis of the analysis of 267 CC samples, we noted 27% of CCs express genes which predict for embryos able to achieve pregnancy, 42% of CCs did not, 31% of CCs showing gene expression for early arrest of embryo development.
Selected candidate biomarkers are listed in the below Table B. Table B presents the set of genes in cumulus cells were able to predict different clinical conditions: (A) pregnancy, (B) absence of pregnancy and (C) early embryo arrest.

TABLE B

set of predictive genes

Gene		Gene
Symbol	Gene name	ID

A: genes whose overexpressions are predictive of pregnancy

PCK1	phosphoenolpyruvate carboxykinase 1 (soluble)	5105
ADPRH	ADP-ribosylarginine hydrolase	141
CABP4	calcium binding protein 4	57010
SLAMF6	SLAM family member 6	114836
CAMTA1	calmodulin binding transcription activator 1	23261
CSPG2	Chondroitin sulfate proteoglycan 2 (versican)	1462
PRF1	perforin 1 (pore forming protein)	5551

B: genes whose overexpressions are predictive of embryos unable to implant

FOSB	FBJ murine osteosarcoma viral oncogene homolog B	2354
NLGN2	neuroligin 2	57555
PDE5A	phosphodiesterase 5A, cGMP-specific	8654
PLA2G5	phospholipase A2, group V	5322
GPC6	glypican 6	10082
EGR3	early growth response 3	1960

C: genes whose overexpressions are predictive of early embryo arrest

NFIB	nuclear factor I/B	4781
NFIC	nuclear factor I/C (CCAAT-binding transcription factor)	4782
IGF1R	insulin-like growth factor 1 receptor	3480
G0S2	G0/G1 switch 2	50486
GIRK5	glutamate receptor, ionotropie, kainate 5	2901
RBMS1	RNA binding motif, single stranded interacting protein 1	5937

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Claims

1-4. (canceled)

5. A kit, comprising:

means for measuring the expression level of genes in a cumulus cell surrounding an oocyte or an embryo, wherein said means for measuring are selective reagents selected from the group consisting of probes, primers, and binding partners which are specific for each of the following 45 genes: WNT6, LRCH4, PAX8, CABP4, PDE5A, BCL2L11, PCK1, TCF20, SLAMF6, EPOR, CACNG6, NLRP1, PECAMI, NOS1, ATF3, KRTAP8, GRIK5, SLC24A3, SLC5A12, SLCA10A2, SLCO1A2, SLC25A5, MG29, NLGN2, PRKACA, FOSB, SIAT6, LOXL2, PRFI, ADPRH, APBB3, EGR3, CNR2, IFITM1, PLA2G5, CAMTA1, SOX4, NFIB, NFIC, RBMSI, G0S2, FAT3, SLC40A1, GPC6 and IGFIR.

6. The kit according to claim 5 further comprising:

a non-transient storage medium containing stored reference values for a control sample having cumulus cells associated with an oocyte or an embryo wherein said reference values include expression levels for said control for each of said 45 genes; and

means for comparing each of said stored reference values to measured values obtained with said means for measuring for each of said 45 genes.

7. The kit according to claim 5, wherein the selective reagents are hybridization probes and wherein said hybridization probes are nucleic acids which are suitable for hybridizing to mRNA of WNT6, LRCH4, PAX8, CABP4, PDE5A, BCL2L11, PCK1, TCF20, SLAMF6, EPOR, CACNG6, NLRP1, PECAM1, NOS1, ATF3, KRTAP8, GRIK5, SLC24A3, SLC5A12, SLCA10A2, SLCO1A2, SLC25A5, MG29, NLGN2, PRKACA, FOSB, SIAT6, LOXL2, PRF1, ADPRH, APBB3, EGR3, CNR2, IFITMI, PLA2G5, CAMTAI, SOX4, NFIB, NFIC, RBMS1, G0S2, FAT3, SLC40A1, GPC6 and IGFIR under high stringency hybridization conditions.

8. The kit according to claim 7 wherein the hybridization probes are chemically attached to a substrate.

9. The kit according to claim 8 wherein the kit comprises a DNA chip comprising said hybridization probes chemically attached to a solid support.

10. The kit according to claim 5, wherein the selective reagents are amplification primers and wherein said amplification primers are single stranded nucleic acids from 10 to 25 nucleotides which are suitable for hybridizing to mRNA of WNT6, LRCH4, PAX8, CABP4, PDE5A, BCL2L11, PCK1, TCF20, SLAMF6, EPOR, CACNG6, NLRP1, PECAMI, NOSI, ATF3, KRTAP8, GRIK5, SLC24A3, SLC5A12, SLCA10A2, SLCO1A2, SLC25A5, MG29, NLGN2, PRKACA, FOSB, SIAM, LOXL2, PRF1, ADPRH, APBB3, EGR3, CNR2, IFITM1, PLA2G5, CAMTA1, SOX4, NFIB, NFIC, RBMSI, G0S2, FAT3, SLC40A1, GPC6 and IGFIR under high stringency hybridization conditions.

11. The kit according to claim 10 wherein the kit comprises reagents for a quantitative or semi-quantitative RT-PCR.

12. The kit according to claim 5, wherein the selective reagents are binding partners and wherein said binding partners are capable of selectively interacting with the proteins WNT6, LRCH4, PAX8, CABP4, PDE5A, BCL2L11, PCK1, TCF20, SLAMF6, EPOR, CACNG6, NLRP1, PECAM1, NOS1, ATF3, KRTAP8, GRIK5, SLC24A3, SLC5A12, SLCA10A2, SLCO1A2, SLC25A5, MG29, NLGN2, PRKACA, FOSB, SIAT6, LOXL2, PRF1, ADPRH, APBB3, EGR3, CNR2, IFITMI, PLA2G5, CAMTA1, SOX4, NFIB, NFIC, RBMS1, G0S2, FAT3, SLC40A1, GPC6 and IGFIR.

13. The kit according to claim 12 wherein the binding partner is antibody.

14. The kit according to claim 13 wherein the kit comprises reagents for an ELISA.