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WO2017011876A1 - Biomarqueurs glycoformes - Google Patents

Biomarqueurs glycoformes Download PDF

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Publication number
WO2017011876A1
WO2017011876A1 PCT/AU2016/050648 AU2016050648W WO2017011876A1 WO 2017011876 A1 WO2017011876 A1 WO 2017011876A1 AU 2016050648 W AU2016050648 W AU 2016050648W WO 2017011876 A1 WO2017011876 A1 WO 2017011876A1
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Prior art keywords
lectin
protein
pair
glycoform
endometrial
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PCT/AU2016/050648
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English (en)
Inventor
Tracey A. EDGELL
Lois Salamonsen
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Prince Henry's Institute Of Medical Research Trading As The Hudson Institute Of Medical Research
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Priority claimed from AU2015902891A external-priority patent/AU2015902891A0/en
Application filed by Prince Henry's Institute Of Medical Research Trading As The Hudson Institute Of Medical Research filed Critical Prince Henry's Institute Of Medical Research Trading As The Hudson Institute Of Medical Research
Publication of WO2017011876A1 publication Critical patent/WO2017011876A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4724—Lectins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/02—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates involving antibodies to sugar part of glycoproteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/367—Infertility, e.g. sperm disorder, ovulatory dysfunction

Definitions

  • the present invention relates to an assay for determining mammalian endometrial or uterine receptivity for an embryo in order to facilitate successful implantation of the embryo by natural or artificial means.
  • the assay further discriminates between fertile and infertile mammalian females at particular secretory phases.
  • Proteome Res 8: 1099-1103 Some of these investigations employ differential display studies of either proliferative and secretory phases (Chen et al (2009) J Proteome Res 8: 2032-2044; DeSouza et al (2005) Proteomics 5: 270-281; Dominguez et al (2009) Hum. Reprod 24: 2607-2617; Li et al (2011) Fertil Steril 95: 1161-1163; Rai et al (2010) Proteomics Clin Appl 4: 48-59) or direct comparison of fertile and infertile cohorts (Hannan et al (2010) J Proteome Res 9: 6256-6264).
  • Dysregulated protein forms can occur by any number of means including alterations in amino acid sequence or post translational changes.
  • quantitation of a single dysregulated protein is problematic.
  • One reason for this is that antibodies frequently are incapable of distinguishing between different post translationally modified forms of the same protein.
  • the alterative is to use an array map or mass-spectrometric analysis which requires specialized equipment and skills, is costly and requires lengthy time frames.
  • the present invention teaches the development of an assay to identify and validate markers of uterine receptivity for an embryo.
  • Reference to "uterine receptivity” and “endometrial receptivity” are used interchangeably herein to determine the state of a uterus most receptive to successful implantation of a healthy embryo by either natural or by assisted reproduction means.
  • the assay samples uterine lavage for particular glycoforms of one or more proteins indicative of endometrial (or uterine) receptivity.
  • a multiplex endometrial protein capture antibody-endometrial protein-lectin assay is taught herein to detect particular glycoforms of endometrial proteins in uterine lavage samples.
  • Conventional sandwich ELISAs use a protein-specific primary capture antibody combined with a secondary antibody carrying a detectable label such as horseradish peroxidase or biotin.
  • the secondary antibody is replaced by a lectin which provides a means to identify whether a specific glycoform is bound to the primary antibody.
  • the lectin forms a glycoconjugate between the protein glycoform and the lectin.
  • the present invention provides an assay to identify and validate glycoform biomarkers in endometrial lavage which are indicative of a receptive or non-receptive uterine environment for successful natural or assisted implantation of an embryo to or near to term.
  • the glycoforms can also discriminate between fertile and infertile female subjects and particular secretory phase.
  • the protein-lectin pairs or glycoconjugates are identified which discriminate fertile from infertile women during mid-secretory phase of a natural cycle and those which discriminate pregnancy outcome among woman undergoing assisted reproduction.
  • one aspect of the present invention provides an assay to identify an endometrial protein glycoform biomarker in uterine lavage from a female mammal which is indicative of a level of receptivity of an endometrium for a likely successful or likely unsuccessful embryo implantation, the assay comprising selecting an endometrial protein having a glycoform which specifically binds to a lectin forming an endometrial protein- lectin binding pair, capturing the protein on a solid support using an immobilized primary antibody specific for the protein and then detecting the glycoform of the captured protein using the lectin specific for the glycoform wherein the lectin is labeled with a reporter molecule capable of being detected or binding a labeled antibody specific for the lectin which antibody is labeled with a reporter molecule capable of being detected.
  • the implantation may be natural or assisted (e.g. by in vitro fertilization [IVF]).
  • Another aspect contemplates the use of a endometrial protein-lectin binding pair, wherein the lectin binds to a specific glycoform of the endometrial protein in the manufacture of an assay to detect uterine receptivity or otherwise for an embryo implant in a female mammal.
  • the present assay applies to human female subjects as well as non-human mammalian female animals. Hence, the assay has human clinical and veterinary applications.
  • Yet another aspect herein enables a method of assisted reproduction a method of assisted reproduction in a female mammal the method comprising selecting an endometrial protein having a glycoform which specifically binds to a lectin forming a protein-lectin pair, capturing the protein on a solid support using an immobilized primary antibody specific for the protein and then detecting the glycoform of the captured protein using the lectin specific for the glycoform wherein the lectin is labeled with a reporter molecule capable of being detected or binding a labeled antibody specific for the lectin which antibody is labeled with a reporter molecule capable of being detected wherein the presence or absence or level of the protein-lectin pair is indicative of whether an embryo implant should proceed.
  • a method of determining a female mammal that is likely to fall pregnant comprising selecting an endometrial protein having a glycoform which specifically binds to a lectin forming a protein-lectin pair, capturing the protein on a solid support using an immobilized primary antibody specific for the protein and then detecting the glycoform of the captured protein using the lectin specific for the glycoform wherein the lectin is labeled with a reporter molecule capable of being detected or binding a labeled antibody specific for the lectin which antibody is labeled with a reporter molecule capable of being detected wherein the presence or absence or level of the protein-lectin pair is indicative of a receptive endometrial for pregnancy.
  • Endometrial proteins contemplated herein are defined in the subject specification and are referred to by the abbreviations ORMl, MUC5B, A2M-like (A2M-L) protein and TAFI (Table 1).
  • the present invention extends to non-human equivalents of these proteins if they do not exist in such mammal.
  • other potential proteins of interest are listed in Tables 19-23 of Example 9.
  • Lectin abbreviations used herein include CONA, DBA, DSL, ECL, GSLI, GSLII, JAC, LEL, PSA, RCA, SBA, SJA, STL, ULEX, VVA and WGA (Table 2).
  • Particular protein-lectin biding pairs include ORMl -DBA, ORM1-CONA, A2M- L-WGA, glycodelin-RCAI, glycodelin-DBA TAFI-DBA and MUC5B-CONA.
  • TAFI Table 2 Thrombin activatable fibrinolysis inhibitor TAFI Table 2
  • Figure 2 is a graphical representation of pooled lavage samples from early secretory (A), infertile early secretory (B), fertile mid-secretory (C) and infertile mid- secretory (D) run on 12% w/v SDS-PAGE and stained with coomassie (Left). The samples were subject to western blot using DBA lectin (Right).
  • Figure 3 is a graphical representation of lavage samples collected during the mid- secretory phase of women with proven fertility (F-MS) and those with known infertility (IF-MS) and assayed for alpha-2-macroglobulin (Luminex assay). Results for each group are presented as mean +/- standard deviation. No significant difference was found.
  • Figures 4A to C are graphical representations of uterine lavage analyzed for the concentrations of three protein glycoforms.
  • the women were classified according to cycle stage, early secretory (es) and mid-secretory (ms), and fertility status, fertile (F) and primary infertile (pif). Results were analyzed by Kruskal-Wallis between groups of women.
  • Figures 5A to D are graphical representations showing concentrations of CONA reactive glycoforms of ORMl and MUC5B analyzed in uterine lavage samples collected from naturally cycling fertile and primary infertile women (left side) during early (ES) and mid-secretory (MS) phases, and from IVF treated infertile women at oocyte collection human chorionic gonadotropin + 2 days (hCG+2). IVF stimulated women were grouped according to outcome of pregnancy (anta P val) or No pregnancy (ana NP val) following fresh embryo transfer in the same cycle. Results are expressed as Flurescence Intensity (FI). Comparison was made between groups using Mann -Whitney analysis.
  • Figure 6 is a photographic representation of a protein gel image from SDS TGX stainfree gel flushing pools from proliferative fertile, proliferative infertile, secretory fertile and secretory infertile (lanes 1-4, respectively) and equal volume loading (lanes 5-8, respectively).
  • the present invention is predicated in part on the identification of endometrial proteins having a particular glycosylation pattern referred to herein as a "glycoform".
  • the presence or absence or level or a particular endometrial protein or the ratio of levels of two or more endometrial proteins is indicative of the likelihood or otherwise of a successful embryo implantation whether by natural or assisted reproduction means.
  • the glycoform is detected by a lectin which binds to a particular glycosylation pattern on the protein of interest to form a glycoconjugate.
  • an endometrial protein-lectin binding pair or glycoconjugate which is the lectin which binds to a particular glycoform of a selected protein. Shortened expressions such as "protein-lectin”, “protein- lectin pair” and “glycoconjugate” have the same meaning as "endometrial protein-lectin binding pair".
  • an assay to identify an endometrial glycoform biomarker in uterine lavage from a female mammal which is indicative of a level of receptivity of an endometrium for a likely successful or likely unsuccessful embryo implantation comprising selecting an endometrial protein having a glycoform which specifically binds to a lectin forming an endometrial protein-lectin binding pair, capturing the protein on a solid support using an immobilized primary antibody specific for the protein and then detecting the glycoform of the captured protein using the lectin specific for the glycoform wherein the lectin is labeled with a reporter molecule capable of being detected or binding a labeled antibody specific for the lectin which antibody is labeled with a reporter molecule capable of being detected.
  • a method of determining a female mammal that is likely to fall pregnant comprising selecting an endometrial protein having a glycoform which specifically binds to a lectin forming a protein-lectin pair, capturing the protein on a solid support using an immobilized primary antibody specific for the protein and then detecting the glycoform of the captured protein using the lectin specific for the glycoform wherein the lectin is labeled with a reporter molecule capable of being detected or binding a labeled antibody specific for the lectin which antibody is labeled with a reporter molecule capable of being detected wherein the presence or absence or level of the protein-lectin pair is indicative of a receptive endometrial for pregnancy.
  • the subject assay is particularly exemplified with respect to human females
  • the concept of the present invention equally applies to assessing the receptivity of any non-human female mammal undergoing assisted reproduction or natural cycling.
  • non-human mammals include horses, cattle, sheep, pigs, deer and zoo-housed mammals including those endangered such as gorillas, monkeys, orangutans and the Kenyan devil.
  • the present assay has both human clinical and veterinary applications.
  • the assay is also useful in the monitoring of natural cycles in mammalian females such as human female subjects.
  • the assay is used to assess the receptivity of a human female.
  • an assay to identify an endometrial protein glycoform biomarker in uterine lavage from a human female which is indicative of a level of receptivity of an endometrium for a likely successful or likely unsuccessful embryo implantation comprising selecting an endometrial protein having a glycoform which specifically binds to a lectin forming an endometrial protein-lectin binding pair, capturing the protein on a solid support using an immobilized primary antibody specific for the protein and then detecting the glycoform of the captured protein using the lectin specific for the glycoform wherein the lectin is labeled with a reporter molecule capable of being detected or binding a labeled antibody specific for the lectin which antibody is labeled with a reporter molecule capable of being detected.
  • a method of determining a human female that is likely to fall pregnant comprising selecting an endometrial protein having a glycoform which specifically binds to a lectin forming a protein-lectin pair, capturing the protein on a solid support using an immobilized primary antibody specific for the protein and then detecting the glycoform of the captured protein using the lectin specific for the glycoform wherein the lectin is labeled with a reporter molecule capable of being detected or binding a labeled antibody specific for the lectin which antibody is labeled with a reporter molecule capable of being detected wherein the presence or absence or level of the protein- lectin pair is indicative of a receptive endometrial for pregnancy.
  • the present invention provides:
  • the endometrial protein or ratios of levels of two or more endometrial proteins predicts a receptive or non-receptive uterine environment for successful embryo implantation by natural or artificial means;
  • the endometrial protein comprises a particular glycosylation pattern referred to herein as a glycoform
  • a lectin which specifically binds to that glycoform to form a glycoconjugate enabling detection and/or quantification of the presence, absence or level of one protein or ratio of two or more proteins enabling a predication to be made on the likelihood or otherwise of a successful embryo implantation.
  • a female mammal includes a human female and a non -human female mammal.
  • a "human female” may also be referred to herein as a " woman”.
  • the female mammal is of baby bearing age but the assay may also be useful in assessing menopausal changes and hormonal changes, especially those changes leading to a predisposition to a uterine cancer.
  • the sample is referred to as a "uterine lavage” or "endometrial lavage” and both terms may be used interchangeably to assess uterine receptivity for embryo implantation.
  • lavage is not to imply any limitation as to the means of obtaining a uterine sample which includes an endometrial sample. Conveniently, a uterine lavage is the least invasive.
  • biological sample may also be applied to the uterine/endometrial sample.
  • the assay includes micro-arrays, macro-arrays and nano-arrays on planar or spherical solid supports.
  • a “sample” includes but is not limited to from 0.5 ⁇ to 50 ml.
  • Successessful means an embryo which goes to term resulting in a live birth whether natural or by caesarean section as well as the embryo developing to an age where a baby can be delivered pre-term, generally by caesarean section.
  • the "likeliness" or “likelihood” of a successful implantation is relative but factors such as health of the embryo, physical condition of the female subject and other factors not necessarily related to the developing fetus do affect the outcome.
  • the implantation procedure does not adversely effect the embryo or endometrium and the female subject remains in a healthy state
  • the selected endometrial protein glycoform can successfully discriminate between a fertile female from a non-fertile female in early, mid or late secretory phase.
  • pregnancy outcome can be predicted with an at least 75% success rate.
  • At least 75% means at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%). This applies to natural and assisted reproduction.
  • the endometrial protein can be referred to as glycoform, glycomarker, biomarker, glycoform biomarker, predictor, marker, indicator or any such term applying to a protein present in endometrial lavage.
  • glycoform glycomarker, biomarker, glycoform biomarker, predictor, marker, indicator or any such term applying to a protein present in endometrial lavage.
  • glycoconjugate applies to a protein-lectin bonded pair.
  • the protein need not solely reside in the endometrial lavage but its presence, absence or level or its ratio of levels with at least one other protein needs to be correlated with uterine receptivity or non-receptivity for embryo implantation.
  • endometrial proteins include but are not limited to orosmucoid 1 (ORM1), mucin 5B (MUC5B), glycodelin, alpha-2-macroglobulin (A2M)-like (A2M-L) protein and thrombin activatable fibrinolysis inhibitor (TAFI).
  • ORM1 orosmucoid 1
  • MUC5B mucin 5B
  • A2M alpha-2-macroglobulin
  • A2M-L alpha-2-macroglobulin-like protein
  • TAFI thrombin activatable fibrinolysis inhibitor
  • Suitable lectins include concanavalin A (CONA), dolichos biflorus agglutinin (DBA), datura stramonium lectin (DSL), erythrina cristagalli lectin (ECL), griffonia (bandeiraea) simplicifolia lectin I (GSLI), griffonia (bandeiraea) simplicifolia lectin II (GSLII), jacalin (J AC), lycopersicon esculentum lectin (LEL), pisum sativum agglutinin (PSA), ricinus communis agglutinin (RCA), glucine max agglutinin (SB A), sophora japonica agglutinin (SJA), solanum tuberosum lectin (STL), ulex europaeus agglutinin I (ULEX), vicia villosa agglutinin (VVA) and triticum vulgaris
  • Examples of particular endometrial protein-lectin binding pairs include ORM1- DBA, ORMl-CONA, A2M-L-WGA, glycodelin-RCAl, glycodelin-DBA, TAFI-DBA and MUC5B-CONA.
  • the endometrial protein-lectin binding pairs are A2M- L-WGA, ORM1-DBA and glycodelin-RCAl .
  • an assay to identify an endometrial protein glycoform biomarker in uterine lavage form a female mammal which is indicative of a level of receptivity of an endometrium for a likely successful or likely unsuccessful embryo implantation comprising selecting an endometrial protein from the group consisting of ORM1, MUC5B, glycodelin, A2M-like protein and TAFI from a human female or their equivalent in a non-human mammal having a glycoform which specifically binds to a lectin selected from the group consisting of CONA, DBA, DSL, ECL, GSLI, GSLII, JAC, LEL, PSA, RCA, SBA, SJA, STL, ULEX, VVA and WGA forming an endometrial protein-lectin binding pair, capturing the protein on a solid support using an immobilized primary antibody specific for the protein and then detecting the glycoform of the captured protein using the lectin specific for the glyco
  • the protein is selected from A2M-like protein, ORMl and glycodelin and the corresponding lectin specific for the particular glycoform is WGA, DBA and RCA1, respectively.
  • Monoclonal or polyclonal antibodies may be used as the capture antibody for the endometrial protein or if needed the "tertiary" antibody.
  • a tertiary antibody is used to bind to a lectin portion of a glycoconjugate.
  • the use of monoclonal antibodies in an immunoassay is particularly useful because of the ability to produce them in large quantities and the homogeneity of the product.
  • the preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.
  • results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of protein. This generalized technique is well known to those skilled in the art as would be any of a number of variations.
  • a "tertiary antibody" may also be used to detect the lectin if the lectin is not labelled.
  • a primary antibody having specificity for the instant protein is either covalently or passively bound to a solid surface.
  • the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, spheres, discs of microplates, or any other surface suitable for conducting an immunoassay. Microspheres are particularly useful.
  • the binding processes are well known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the solid phase-antibody complex is washed in preparation for the test sample.
  • An aliquot of the lavage to be tested is then added to the solid phase antibody and incubated for a period of time sufficient (e.g. 2-120 minutes or where more convenient, overnight) and under suitable conditions (e.g. for about 20°C to about 40°C) to allow binding of any subunit present in the lavage.
  • the reaction vessel is washed and dried and incubated with a lectin specific for the glycoform of the protein.
  • the lectin is generally linked to a reporter molecule which is used to indicate the binding of the protein to the primary antibody.
  • reporter molecule as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative.
  • reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules. Examples of suitable fluorophores well known.
  • an enzyme is conjugated to the lectin, generally by means of glutaraldehyde or periodate.
  • glutaraldehyde or periodate As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta- galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase.
  • fluorogenic substrates which yield a fluorescent product rather than the chromogenic substrates noted above.
  • the enzyme-labeled lectin is added to the immobilized antibody- protein complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-protein- lectin. The substrate will react with the enzyme linked to the lectin, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of protein which was present in the sample.
  • the present disclosure extends to a substantially simultaneous assay.
  • fluorescent compounds such as fluorescein and rhodamine
  • fluorescein and rhodamine may be chemically coupled to lectins without altering their binding capacity.
  • the flurochrome-labeled lectin When activated by illumination with light of a particular wavelength, the flurochrome-labeled lectin adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope.
  • the fluorescent labeled lectin is allowed to bind to the first antibody-protein complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the protein of interest.
  • Immunofluorescence and enzyme immunoassay techniques are both very well established in the art. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
  • the assay enabled herein may be automated or semi -automated for high throughput screening or for screening for a protein glycoforms from the one or multiple subjects.
  • the automation is conveniently controlled by computer software.
  • the present disclosure further contemplates therefore web-based and non-web- based systems where data on the receptivity of a subject are provided by a client server or other architecture platform to a central processor which analyses and compares to a control and optionally considers other information such as patient age, weight and other medical conditions and then provides a report, such as, for example, a risk factor for embryo implantation failure.
  • a client server or other architecture platform to a central processor which analyses and compares to a control and optionally considers other information such as patient age, weight and other medical conditions and then provides a report, such as, for example, a risk factor for embryo implantation failure.
  • business method is provided whereby uterine lavage is collected in transportable tubes which is then analyzed for protein glycoforms at a defined location and the results then sent in the form of an electronic report via a client server or other architecture platform to a clinical care provider.
  • knowledge-based computer software and hardware also form part of the present disclosure. This facilitates clinical care to ascertain whether a subject has a degree of endometrial receptivity sufficient to expect a likely successful pregnancy.
  • kits for use with the methods described above In a further embodiment, the present disclosure enables kits for use with the methods described above. In one embodiment, an glycoconjugate kit is contemplated.
  • the immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with or linked to the given protein glycoform, and detectable labels that are associated with or attached to lectin. Exemplary lectins are listed in Table 2.
  • kits may be packaged either in aqueous media or in lyophilized form.
  • the container means of any of the kits generally includes at least one vial, test tube, flask, bottle, syringe or other container means, into which the testing lavage is placed, and generally, suitably aliquoted. Where a lectin is provided, the kit will also generally contain a second, third or other additional container into which this lectin may be placed.
  • the kits taught by the present disclosure also typically include a means for immobilizes the protein of interest and any other reagents.
  • the present specification is also enabled for the use of an endometrial protein- lectin binding pair, wherein the lectin binds to a specific glycoform of the endometrial protein in the manufacture of an assay to detect uterine receptivity or otherwise for an embryo implant in a female mammal such as a human female.
  • the assay may be used in a method of assisted reproduction in a female mammal the method comprising selecting an endometrial protein having a glycoform which specifically binds to a lectin forming a protein-lectin pair, capturing the protein on a solid support using an immobilized primary antibody specific for the protein and then detecting the glycoform of the captured protein using the lectin specific for the glycoform wherein the lectin is labeled with a reporter molecule capable of being detected or binding a labeled antibody specific for said lectin which antibody is labeled with a reporter molecule capable of being detected wherein the presence or absence of the protein-lectin pair is indicative of whether an embryo implant should proceed.
  • the female mammal is a human female.
  • the assay is used to predict receptivity for a natural pregnancy.
  • a primary antibody to a particular protein identified in uterine lavage is immobilized to a solid support.
  • the solid support may be any support including a microsphere, microparticle or the side of a reaction vessel.
  • the primary antibody is immobilized via its constant domains in the Fc region.
  • a lavage sample is brought into contact with the immobilized primary antibody for a time and under conditions sufficient for a protein to which the antibody is specific binds. Unbound material and superfluent lavage fluid is washed away. A selected lectin specific for a glycoform of the captured protein is then added.
  • the lectin is labeled with a detectable marker or label such as streptavidin, horseradish peroxidase or detection is via a tertiary antibody specific for the captured lectin.
  • a detectable marker or label such as streptavidin, horseradish peroxidase or detection is via a tertiary antibody specific for the captured lectin.
  • microspheres are transferred to a microcentrifuge tube and pelleted at 8000 g. The supernatant is then removed and the beads suspended in 100 mm sodium phosphate, pH 6.2, by Vortex agitation and sonication. Conjugation reagents (e.g., sulfo- HS) and EDC are added to the microspheres and incubated with gentle mixing followed by centrifugation. Supernatant is then removed. The pelleted beads are resuspended in 50 mm MES, pH 5.0 and washed twice in the same buffer by repeated pelleting and re-suspension. The selected antibody is added to the microspheres and incubated with mixing. To optimize coupling, initially a titration of various antibody contractions is used.
  • microspheres are pelleted and PBS/Tween 20 buffer added to block further reaction sites. Antibody binding is confirmed by incubating the microspheres with serial dilutions of a phycoerythrin anti-species IgG detection antibody in the wells of a pre- wetted filtration plate. After suctioning off the excess liquid and washing away inbound detection antibody, the microspheres are resuspended and screened for the IgG detection antibody.
  • Preliminary assessment of proteins of interest is made using lavage samples from individuals with proven fertility or infertility. In each group, approximately 5-6 samples collected at early/mid secretory phase are used. The samples are each mixed with the microsphere beads conjugated with antibody to the protein of interest in a prewetted 96- well filtration plate and incubated. Replicate wells of each sample/microsphere mixture are prepared to permit secondary detection with both a biotin conjugated antibody to the protein, and with the identifying lectin also biotinylated. Following incubation of microspheres and sample, the liquid is suctioned off, and the wells washed by three applications of wash buffer, with suctioning in between.
  • the secondary agent (antibody- biotin or lectin-biotin) is then applied and incubated. After further washing, the detection reagent, streptavidin-phycoerthythrin is added to all wells. Following incubation and exhaustive washing, the microspheres are resuspended in Bioplex sheath fluid and analysed on a Bioplex-200 instrument. The detected signal quantified as fluorescence intensity will be determined for each well. A blank well for each microsphere/secondary reagent in which sample is substituted with buffer is used to assess any background binding occurring between the secondary reagent and the immobilized capture antibody.
  • ICPL-4 The protein from F and IF pools captured using two different lectins is labeled using ICPL-4 reagent (Kellermann (2008) Methods mol boil 424: 113-123). Using ICPL-4 it is possible to label four proteomic states i.e. 4 samples for simultaneous quantitation (Lottspeich and Kellermann (2011) Methods Mol Biol 753: 55-64; Schmidt et al (2005) Proteomic S 5: 4-15). ICPL provides a top down approach labeling the protein prior to separation and enzymatic digestion. ICPL contains four isotopically labeled variants which respectively add a recognisable mass-shift relative to the native isotopic form. Thus, with each of four samples labeled with a unique variant, they may be combined for analysis.
  • each isolate is labeled it is separated using OFFGEL separation.
  • Conventional 2D electrophoresis performs an initial isoelectric focussing (pi separation) with a secondary molecular weight separation of a complex protein mixture.
  • OFFGEL has been demonstrated to provide improved proteomic coverage.
  • the initial isoelectric separation is performed in the liquid phase.
  • a rehydrated IPG strip is sealed within the well frame and the diluted solution (ICPL-4 labeled combination of four lectin isolates) added equally to all wells and sealed to prevent evaporation.
  • High voltage is applied to the IPG strip and the proteins migrate until they reach a position where the pH matches their pi. After separation the proteins remain within their respective wells until transferred for further analysis.
  • each well is further separated by HPLC before the proteins are digested with Glu-C and analyzed using maldi-TOF spectrometry.
  • the ICPLquant software (Brunner et al (2010) Proeomics 10: 315-326) is used to analyze the obtained spectra to identify the proteins present but also to make a quantitative comparison of each ICPL label for each identified protein, thus providing quantitative detail as to the abundance of a glycoform.
  • it will identify glycoforms significantly dysregulated between the paired F and IF isolates for each lectin.
  • a preliminary histochemical screen with a number of lectins on endometrial tissue taken at different phases of the menstrual cycle confirms that some lectins demonstrate a phase dependent binding to endometrial tissue (Figure 1).
  • the patient cohorts were selected to provide a detailed assessment of the ability of protein glycoform assays to determine endometrial receptivity.
  • a larger sample set of 40 subjects comprised:
  • the inclusion of the early secretory phase fertile women within this sample set provides an indication whether the protein glycoform expression is mid-secretory phase receptive specific, given that during the early secretory phase it is expected that the endometrium will be non-receptive. Further the inclusion of early secretory phase infertile women would indicate if there is a dys-synchrony in their endometrial receptivity.
  • Multivariate modelling of the three assays was performed using WEKA software, with the mid-secretory data input. Results with the mid-secretory fertile and infertile data produced 100% correct classification, and an AUC of 1.000. However, no test data set is available; given it is the infertile mid-secretory women who differ in expression levels of the three assays, the use of the early secretory data cannot be used as a test set of non- receptivity.
  • Each pooled was assayed for protein concentration using BCA protein assay. Each sample was assayed in duplicate at three dilution (neat, 1 :4, 1 :9), and the assay repeated by two operators. Mean protein concentration was determined for each pool, and the results are shown in Table 10.
  • Protein determined using BCA assay for flushings pools are shown in ug/ml. Values shown are means from duplicate assays performed at three sample dilutions, and replicated by two operators.
  • Table 11 summarizes the determined optimal concentrations of lectin and inhibitor sugar used in immunohistochemistry studies. The appropriate dilution of individual lectins for immunohistochemistry was determined in initial studies. For each lectin an appropriate neutralizing sugar and its' concentration required to achieve full inhibition of lectin binding to tissue for use as a negative control was also determined.
  • Table 12 summarizes the optimal concentrations for western blotting with each lectin. The appropriate dilution of individual lectins for western blotting was determined in initial studies.
  • FIG. 6 A representative protein gel image is shown in Figure 6. The gel is loaded in similar manner as was performed for each western blot, thus the four sample pools are shown at equal protein and equal volume loading. [0102] IHC studies show demonstrable CONA recognition throughout the cycle, however, it is seen to elevate in the mid to late secretory phase in fertile women. This elevation is less clear in the infertile cohort.
  • DBA staining within the luminal epithelia was sparse or zero in the proliferative phase tissues of both fertile and infertile individuals. The staining increased throughout the secretory phase and was apically positioned within the cells. The infertile women displayed greater expression, and this was indeed also true within the glandular epithelium where highly intense staining throughout the majority of glands was visible by the mid secretory phase, and visible secretions within the glands also noted. Western analysis though reactivity was weak, revealed two bands which appeared uniquely within the mid- secretory infertile pool.
  • GSLII appears to bind more strongly to secretory phase tissue from fertile women, compared with proliferative phase tissue. In addition there is an indication that infertile women showed less reactivity during the secretory phase. However, western analysis showed very poor reactivity with GSLII.
  • LEL binding reaches a maximum in the mid secretory phase of fertile women, evident in both the glandular and luminal epithelium. However there appeared no clear pattern of difference between fertile and infertile women.
  • Western analysis identified two small molecular weight bands (21 and 22kDa) and a larger 70kDa band, prominent in all groups except the Infertile proliferative pool. Additionally, longer exposure identified a 45kDa band visible only in the Infertile proliferative pool.
  • Tissue analysis showed a cyclic expression pattern of SB A binding sugars notably in the glandular epithelium, where in fertile women a trough occurs at the early secretory stage, followed by an increase in the mid secretory phase. These is a suggestion that the infertile cohort may show a somewhat reduced expression throughout the cycle. In western analysis of sample pools there was no clearly visible difference in binding of the SBA lectin.
  • STL showed a very uniform level of staining of tissues regardless of stage or fertility status, though actual signal strength was quite strong. In western analysis however despite a lengthy imaging of 30 minutes very little signal could be observed, perhaps indicative that the STL recognized sugars are primarily on non-secreted proteins within the tissue.
  • ULEX showed a very disparate binding to tissues throughout the cycle with both high and low binding samples in all stages of the cycle. ULEX blotting did not show differences between the pooled groups. The differences observed between lanes, appear to be dependent on protein loading as they are not evident in lanes where equal protein is loaded.
  • This Example relates to the proteomic identification of protein glycoforms showing unique or dysregulated expression in uterine lavage samples between fertile and infertile women in the early-midsecretory phase.
  • CONA Three lectins, CONA, RCA-1 and WGA, that capture glycoproteins were selected for further investigation based on the data the previous Examples. These were applied to pooled secretory phase lavage from fertile and infertile women. Subsequently CONA analyses were repeated using individual samples. This allowed more detailed analysis of early and mid-secretory phases individually.
  • This process isolates and identifies only the peptide region carrying the actual glycosylation of interest, allowing analysis of changes to specific glyosylation sites.
  • this method can provide for future sequential lectin absorptions as only peptides, not all regions of the protein, are selectively bound at each stage.
  • Protein lists are shown in Tables 19 to 23. Identifications are color coded as shown:
  • mimsm BIBLIOGRAPHY mimsm BIBLIOGRAPHY:

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Abstract

La présente invention concerne un dosage permettant de déterminer la réceptivité de l'endomètre ou de l'utérus d'un mammifère pour un embryon dans le but de faciliter une implantation réussie de l'embryon par des moyens naturels ou artificiels. En outre, le dosage fait une distinction entre des mammifères femelles fertiles et infertiles lors de phases sécrétoires particulières. Le dosage implique la détection de glycoformes protéiques endométriaux particuliers en utilisant à la fois une lectine spécifique et un anticorps spécifique.
PCT/AU2016/050648 2015-07-21 2016-07-21 Biomarqueurs glycoformes WO2017011876A1 (fr)

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EP4027142A4 (fr) * 2019-09-02 2023-09-27 Fujirebio Inc. Méthode de mesure d'une substance de liaison à la lectine, kit de mesure d'une substance de liaison à la lectine et support de capture à utiliser dans cette dernière

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4027142A4 (fr) * 2019-09-02 2023-09-27 Fujirebio Inc. Méthode de mesure d'une substance de liaison à la lectine, kit de mesure d'une substance de liaison à la lectine et support de capture à utiliser dans cette dernière
US12038440B2 (en) 2019-09-02 2024-07-16 Fujirebio Inc. Lectin-binding substance measurement method, lectin-binding substance measurement kit, and capture carrier for use in these
CN112034181A (zh) * 2020-08-31 2020-12-04 西北大学 一种凝集素探针组合在基于尿蛋白糖型鉴定黑叶猴妊娠方面的应用

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