WO2023239935A1 - Détection du facteur intrinsèque (if) gastrique dans des échantillons de fluide - Google Patents

Détection du facteur intrinsèque (if) gastrique dans des échantillons de fluide Download PDF

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Publication number
WO2023239935A1
WO2023239935A1 PCT/US2023/024980 US2023024980W WO2023239935A1 WO 2023239935 A1 WO2023239935 A1 WO 2023239935A1 US 2023024980 W US2023024980 W US 2023024980W WO 2023239935 A1 WO2023239935 A1 WO 2023239935A1
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antibody
binding agent
epitope
sample
composition
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PCT/US2023/024980
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English (en)
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Jonathan Bortz
Eva Greibe
Ebba Nexo
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Xeragenx Llc
Aarhus Universitet
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Publication of WO2023239935A1 publication Critical patent/WO2023239935A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • the present disclosure provides immunological assays and diagnostics for quantification of IF in a fluid sample.
  • IF Gastric Intrinsic Factor
  • the protein Gastric Intrinsic Factor (IF) is produced by parietal cells in the gastric mucosa and secreted into the gastrointestinal tract where it facilitates the intestinal absorption of vitamin B12.
  • IF deficiency can lead to vitamin B12 deficiency which, if left untreated can cause anemia and a spectrum of neurological conditions.
  • Changes in IF level may occur because of genetic factors, infections (for example H. pylori), autoimmune diseases, surgical procedures, atrophic stomach lining and other conditions.
  • infections for example H. pylori
  • autoimmune diseases for example, autoimmune diseases, surgical procedures, atrophic stomach lining and other conditions.
  • patients with the autoimmune disease, pernicious anemia either produce reduced or no IF or have autoantibodies against IF, which results in a reduced or no ability to absorb B12 efficiently.
  • IF is present in gastric juice in concentrations of nmol/L.
  • measuring of the protein in gastric juice is of little clinical use due to difficulties in persuading patients to donate sample material and due to difficulties with standardization of samples collection.
  • IF has never been successfully detected in serum samples and other body fluids.
  • Ability to detect IF in easily accessible fluid samples like serum may greatly facilitate early diagnosis of underlying conditions and improve patient outcomes.
  • One aspect of the present disclosure encompasses a composition, the composition comprising a complex of: (a) an epitope-binding agent that binds -B12 saturated IF (bound-IF) and (b) IF or fragments thereof.
  • the IF is present in a biological fluid sample including but not restricted to blood, plasma, serum, urine, amniotic fluid or gastrointestinal fluid.
  • the epitope-binding agent is an antibody.
  • the epitope-binding agent is obtained from an animal (non-human mammal) immunized with non-denatured bound-IF.
  • the IF used to immunize the animal maybe native IF, recombinant IF or synthetic IF.
  • the bound-IF may be purified from human gastric samples under nondenaturing conditions and in the presence of B12.
  • the bound-IF may be obtained through recombinant expression of IF followed by purification
  • the current disclosure also encompasses a composition comprising an epitope-binding agent that binds bound-IF, wherein the epitope binding protein is produced by a method comprising: (a) purifying IF under non-denaturing conditions in the presence of B12; (b) immunizing an animal with the purified IF, wherein the animal is not a human and can be for example a rabbit, mouse, rat, hamster, or camel.
  • the purified IF can be from humans or from a recombinant or synthetic source.
  • the epitope-binding agent is an antibody or an antigenbinding fragment thereof.
  • epitope-binding agents may include but are not restricted to a polyclonal antibody, a full-length antibody comprising an IgG molecule, a VH, a VL, a CDR, a Fab, a F(ab’)2, a nanobody, a monoclonal antibody or an antigen binding fragment thereof.
  • the compositions provided herein further comprise a detection molecule.
  • the detection molecule is conjugated to the epitope-binding agent.
  • Non-limiting examples of detection molecule include horseradish peroxidase (HRP), radioisotopes, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles and/or ligands, such as biotin.
  • HRP horseradish peroxidase
  • radioisotopes haptens
  • fluorescent labels phosphorescent molecules
  • chemiluminescent molecules chemiluminescent molecules
  • chromophores chromophores
  • luminescent molecules such as photoaffinity molecules
  • photoaffinity molecules such as biotin.
  • the current disclosure also encompasses use of the compositions provided herein for detection and/or quantification of IF or fragments thereof in a sample that may be a biological sample, non-limiting examples of which include but are not restricted to blood, serum, plasma, amniotic fluid, gastrointestinal fluid, tissue sample, cell lysate, biopsy, and stool.
  • a sample may be a biological sample, non-limiting examples of which include but are not restricted to blood, serum, plasma, amniotic fluid, gastrointestinal fluid, tissue sample, cell lysate, biopsy, and stool.
  • the sample may be a non- biological sample.
  • compositions provided herein may be used in for example in an immunological assay, non-limiting example as immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), immunohistochemistry (IHC), immunoprecipitation, and Western blotting.
  • immunological assay non-limiting example as immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), immunohistochemistry (IHC), immunoprecipitation, and Western blotting.
  • compositions and methods provided herein may be used to diagnose a subject who is suspected of having an IF-related disease and/or condition, for example pernicious anemia, megaloblastic anemia, gastritis, atrophic gastritis, peptic ulcers, gastrinoma, gastric tumors, gastric ulcers caused by infections like those caused by H.
  • an IF-related disease and/or condition for example pernicious anemia, megaloblastic anemia, gastritis, atrophic gastritis, peptic ulcers, gastrinoma, gastric tumors, gastric ulcers caused by infections like those caused by H.
  • the subject may have lower than standard production of IF, for instance in anemias, ulcers, genetic disorders linked to mutations in GIF gene. In some aspects, the subject may have higher than standard production of IF, for instance in gastrinomas
  • the current disclosure also encompasses a method of detecting IF in a sample from a subject, the method comprising (a) contacting the sample obtained from the subject with an epitope-binding agent, wherein the epitope-binding agent specifically binds IF or fragments thereof in the sample to comprise an IF-binding agent complex; (b) detecting the IF- binding agent complex in the sample.
  • the sample may be a biological fluid for example blood, plasma, serum, gastrointestinal fluid, amniotic fluid, tissue sample, cell lysate, biopsy, urine and stool.
  • the epitope-binding agent is an antibody for example a polyclonal antibody, a full-length antibody, an IgG, a VH, a VL, a CDR, a Fab, a F(ab’)2, a nanobody, a monoclonal antibody or an antigen binding fragment thereof.
  • the epitope-binding agent may be conjugated to the detection molecule, non-limiting examples of which include horse-radish peroxidase (HRP), radioisotopes, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles and/or ligands, such as biotin.
  • HRP horse-radish peroxidase
  • the methods of detection of IF may be an immunoassay, for example an immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), immunohistochemistry (IHC), immunoprecipitation, or western blotting.
  • ELISA immunosorbent assay
  • FACS fluorescence-activated cell sorting
  • IHC immunohistochemistry
  • western blotting for example an immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), immunohistochemistry (IHC), immunoprecipitation, or western blotting.
  • the detection method can detect less than 0.1 , or less than 0.2, or less than 0.3, or less than 0.4, or less than 0.5, or less than 0.6, or less than 0.7, or less than 0.8, or less than 0.9, or less than 1 , or less than 2, or less than 5, or less than 10, or less than 20, or less than 50, or less than 100 pmol/L of IF and wherein the imprecision of detection is within 5%, or within 10%, or within 15% or within 20% or within 25% of the value.
  • the current disclosure also encompasses methods of diagnosing a subject in need thereof with an IF-related disorder, the method comprising: (a) contacting a fluid sample obtained from the subject in need thereof, wherein the epitope-binding agent specifically binds to IF or fragments thereof in the sample to comprise an IF-binding agent complex; (b) quantifying a level of the IF-binding agent complex; and diagnosing the subject with a gastric-IF related disorder by comparing the level to a level from a healthy subject.
  • Non-limiting examples of IF-related disorders include pernicious anemia, megaloblastic anemia, gastritis, atrophic gastritis, peptic ulcers, gastrinoma, gastric tumors, gastric ulcers caused by infections like those caused by H. pylori, genetic disorders linked to mutations in GIF gene, damages to stomach lining or portions of stomach parietal cells, damages due to bariatric surgery, gastric bypass surgery, gastric problems linked to certain neurological conditions, and certain autoimmune conditions like presence of anti-IF antibodies or anti-stomach parietal cell antibodies.
  • the subject may exhibit no IF production. If some aspects, the subject may have lower than standard production of IF, for instance in anemias, ulcers, genetic disorders linked to mutations in GIF gene. In some aspects, the subject may have higher than standard production of IF, for instance in gastrinomas.
  • the sample may be a biological fluid for example blood, plasma, serum, gastrointestinal fluid, amniotic fluid, tissue sample, cell lysate, biopsy, urine and stool.
  • the epitope-binding agent is an antibody for example a polyclonal antibody, a full-length antibody, an IgG, a VH, a Vi_, a CDR, a Fab, a F(ab’)2, a nanobody, a monoclonal antibody or an antigen binding fragment thereof.
  • the epitope-binding agent may be conjugated to the detection molecule, nonlimiting examples of which include horse-radish peroxidase (HRP), radioisotopes, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles and/or ligands, such as biotin.
  • HRP horse-radish peroxidase
  • radioisotopes haptens
  • fluorescent labels phosphorescent molecules
  • chemiluminescent molecules chemiluminescent molecules
  • chromophores chromophores
  • luminescent molecules chromophores
  • photoaffinity molecules such as biotin
  • the methods of detection of IF may be an immunoassay, for example an immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), immunohistochemistry (IHC), immunoprecipitation, or western blotting.
  • ELISA immunosorbent assay
  • the epitope-binding agent may comprise a detection molecule is selected from a group consisting of horse-radish peroxidase (HRP), radioisotopes, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles and/or ligands, such as biotin.
  • HRP horse-radish peroxidase
  • kits comprising: the compositions provided herein and instructions for use.
  • the kit may also comprise one or more of a detection antibody, reagents for an immunoassay, means to obtain a biological fluid.
  • the current disclosure also encompasses a method of determining the effectiveness of administration of B12 in a subject in need thereof: (a) contacting a fluid sample obtained from the subject in need thereof, wherein the epitope-binding agent binds to bound-IF in the sample to comprise an IF- binding agent complex, (b) quantifying a level of the IF-binding agent complex.
  • FIG. 1 shows the effect of adding B12 to the ELISA buffer, to improve the sensitivity of the assay.
  • FIG. 2 shows the elution profile of size exclusion chromatography of endogenous human IF in serum measured by prototype IF ELISA.
  • X-axis indicates the fraction number.
  • Elution volume for void volume (Vo), human HC, albumin, human TC, and total volume (Vt) are shown by arrows. Results are given in arbitrary units.
  • FIG. 3A-3B provides evaluation of the ELISA for human IF on serum.
  • A is a typical calibration curve for IF in the concentration range of 0.3-50 pmol/L.
  • the mean intra-assay (within plate) imprecision (CV%intra) calculated from each serum pool is indicated.
  • the overall intra- assay imprecision was found to be 6.9%.
  • FIG. 4 provides a graphical representation of the diurnal variation of serum IF in healthy subjects.
  • Serum IF was measured at different time points three days in a row in 20 healthy individuals.
  • the 24-hour clock format is used to indicate time of the day.
  • IF may be released not only into the gastric juice but also into the circulation. This led to the discovery that minute amounts of IF are present in blood and urine samples and can be detected by novel methods provided herein. IF is an important indicator of multiple disease conditions. However, heretofore detection of IF required analysis of gastrointestinal samples that are difficult to obtain. An important and novel aspect of the current disclosure is the confirmation, using the ultra-senstive methods disclosed herein, of a thus far hypothesized small amount of IF in the blood that is retro-secreted by the parietal cells in the stomach of healthy human subjects.
  • the current disclosure encompasses novel and ultra-sensitive methods that were designed to detect IF in the pmol/L range. These novel methods may prove to be extremely useful in clinical settings for quick and reliable detection of a range of IF-related disease conditions.
  • the current disclosure provides immunological methods or immunoassays, for example ELISA (enzyme-linked immunosorbent assay), for detection of IF in blood, plasma, serum, urine, amniotic fluid and other bodily fluids.
  • ELISA enzyme-linked immunosorbent assay
  • An important aspect of the current disclosure is the development of novel epitope-binding agents designed to target B12 saturated samples containing IF and fragments thereof, that make sensitive detection of IF in biological fluids possible.
  • Detailed experimental analysis was conducted to further optimize the methods, determine clinical ranges, standards, and thresholds for developing diagnostics that can be implemented in clinical and non-clinical settings.
  • Statistical analysis was conducted to further determine variations in sample population, thus helping to determine clinically accurate ranges for several disease conditions.
  • novel ELISA based assay methods can be used for quick and easy diagnosis and to inform treatment options for multiple disease conditions including but not limited to pernicious anemia, megaloblastic anemia, gastritis, atrophic gastritis, peptic ulcers, gastrinoma, gastric tumors, gastric ulcers caused by infections like those caused by H. pylori, genetic disorders linked to mutations in GIF gene, damages to stomach lining or portions of stomach parietal cells, damages due to bariatric surgery, gastric bypass surgery, gastric problems linked to certain neurological conditions, and certain autoimmune conditions like presence of anti-IF antibodies or anti-stomach parietal cell antibodies.
  • IF levels may have changes in IF levels, sometimes resulting in reduced or no production of IF and sometimes resulting in increased production.
  • these assay methods are non-invasive and provide strong specificity, very high sensitivity, excellent repeatability, low-cost, and simplicity, thereby offering rapid diagnosis, prevention, and better outcomes for patients.
  • these assays can also be used to predict the outcome of B12 administration in certain patient populations.
  • the current disclosure encompasses diagnostic methods, method of treatment, compositions, and kits for detecting and accurately determining IF levels in biological fluid sample including but not restricted to blood, plasma, serum, urine, amniotic fluid or gastrointestinal fluid using a novel set of antibodies and ELISA based methodologies.
  • the current disclosure encompasses epitope-binding agents that can selectively bind IF or fragments thereof with enough sensitivity and specificity that the bound complex is detectable in human biological samples other than just in gastrointestinal fluids.
  • epitope-binding agent encompasses a protein sequence and variants and fragments thereof, that specifically bind to an epitope on IF to form an IF-binding agent complex. The presence of the complex can be detected by any suitable method; for example, those provided herein.
  • bound-IF refers to IF bound to B12, also refered to as cobalamin or variants thereof including but not restricted to, cobalamin, methyl cobalamin, cyanocobalamin, or vitamin B12.
  • binding of B12 to IF results in structural changes in the molecule that may expose epitopes hitherto unavailable for binding.
  • bound-IF may also refer to a population of IF molecules or variants or fragments thereof, with more than 50%-100% of IF molecules bound to B12.
  • the population of IF molecules may comprise IF molecules or variants or fragments thereof, with 70% or more, or 75% or more, or 80% or more, or 90% or more, or 99% or more of the IF moelcules bound to B12.
  • bound-IF encompasses IF in a non-dentauring liquid medium comprising about 500 pg/mL to about 5000 pg/mL of B12.
  • the non-denaturing liquid medium comprises about 500 pg/mL to about 1000 pg/mL, or about 1000 pg/m L to about 1500 pg/m L, 1500 pg/m L to about 2000 pg/m L, 2000 pg/m L to about 2500 pg/mL, 2500 pg/mL to about 3000 pg/mL, 3000 pg/mL to about 3500 pg/mL, 3500 pg/mL to about 4000 pg/mL, 4000 pg/mL to about 4500 pg/mL, 4500 pg/mL to about 5000 pg/mL of B12.
  • the epitope-binding agents provided herein comprise one or more of antibodies, aptamers, peptides, proteins, polynucleotides, that target IF.
  • the term epitope-binding agent encompasses novel antibodies disclosed herein, that may be obtained by immunization of an animal (not including humans) with non-denatured, B12 saturated IF (bound-IF) and isolating the immunoglobulin fraction of the serum.
  • the IF used to immunize an animal may be derived from a native (human) or non-native recombinant or artificial source under non-denaturing, B12 saturated conditions.
  • the term epitope-binding agent also encompasses immunological molecules comprising one or more amino acid sequences at least about 80% identical to an epitope binding region of these novel antibodies.
  • epitope-binding agent also encompasses compositions comprising sequences corresponding to one or more epitope binding regions of the disclosed antibodies that specifically target bound-IF and/or fragments thereof for example VH, VL, CDRS etc.
  • epitope binding protein also encompasses variants including but not restricted to sequence variants, conjugates with detection molecules etc.
  • antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to polyclonal antibodies, derived monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired epitope-binding activity.
  • the term “antibody” (Ab) includes, without limitation, an immunoglobulin which binds specifically to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • VH heavy chain variable region
  • the heavy chain constant region comprises at least three constant domains, CH1 , CH2 and CH3.
  • Each L chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region comprises one constant domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the H and L chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • the term “antibody” (Ab) also includes, without limitation, any antigen-binding portion of an immunoglobulin which binds specifically to an antigen and is as defined herein.
  • An immunoglobulin can derive from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM.
  • IgG subclasses are also well known to those in the art and include but are not limited to human lgG1 , lgG2, lgG3 and lgG4.
  • “Isotype” refers to the antibody class or subclass (e.g., IgM or lgG1 , respectively) that is encoded by the heavy chain constant region genes.
  • antibody includes, by way of example, both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; wholly synthetic antibodies; and single chain antibodies.
  • a non-human antibody can be humanized by recombinant methods to reduce its immunogenicity in man.
  • the term “antibody” (Ab) also includes an “antigen-binding fragment” or an “antigen-binding portion” or “epitope binding region” of any of the immunoglobulins and includes a monovalent and a divalent fragment or portion, and a single chain antibody.
  • polyclonal antibody describes a composition of different (diverse) antibody molecules which is capable of binding to or reacting with several different specific antigenic determinants on the same or on different antigens.
  • Polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes).
  • polyclonal antibodies against IF may recognize at least one, two, three, or more epitopes on IF.
  • the variability of the binding characteristics of a polyclonal antibody is determined by the so-called variable regions of the polyclonal antibody, in particular in the complementarity determining regions CDR1 , CDR2 and CDR3 regions.
  • a polyclonal antibody may be obtained by separating the target antibody from the antiserum obtained by immunizing an animal for production (for example mouse, rat, rabbit, goat, horse or the like but not humans) with the immunogen according to a general method.
  • the term “monoclonal antibody,” as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. , the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the presently disclosed subject matter may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
  • the current disclosure encompasses monoclonal antibodies comprising one or more epitope-binding sequences that correspond to the epitope binding regions of the polyclonal antibodies provided herein.
  • the monoclonal antibodies may be separately obtained using bound nondenatured IF for selection.
  • an “isolated antibody” refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that binds specifically to IF is substantially free of antibodies that bind specifically to antigens other than the IF). Moreover, an isolated antibody can be substantially free of other cellular material and/or chemicals.
  • an “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.
  • An “antigenbinding portion” or “epitope binding region” of an antibody refers to one or more fragments of an antibody that retain the ability to bind specifically to the antigen bound by the whole antibody.
  • the term “binds” or “binding” or “specific binding” or “specifically binds” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction.
  • Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target.
  • telomere binding or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a Kofor the target of 10" 4 M or lower, alternatively 10" 5 M or lower, alternatively 10" 6 M or lower, alternatively 10" 7 M or lower, alternatively 10" 8 M or lower, alternatively 10" 9 M or lower, alternatively 1O -10 M or lower, alternatively 10" 11 M or lower, alternatively 10 -12 M or lower or a Ko in the range of 10" 4 M to 10" 6 M or 10 -6 M to 10 -10 M or 10" 7 M to 10 -9 M.
  • the epitope binding agent of the current disclosure may have a KD of 10 -1 ° M or lower, or 10 -11 M or lower or 10 -12 M or lower for IF.
  • affinity and KD values are inversely related. A high affinity for an antigen is measured by a low KD value.
  • specific binding refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.
  • the current disclosure also encompasses epitope-binding agents comprising one or more polypeptide sequences that correspond to the epitope binding region or antigen-binding region or antigen-binding fragment of the isolated polyclonal antibodies.
  • the sensitive polyclonal antibodies of the current disclosure can be sequenced to determine the epitope binding regions, variable regions or CDRs that provide high sensitivity.
  • these sensitivity determining CDRs may be incorporated into monoclonal antibodies, synthetic antibodies, recombinant antibodies, antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, Fv), single-chain antibody (scFv), fusion proteins comprising an antibody portion (e.g., chimeric antigen receptor or CAR), humanized antibodies, chimeric antibodies, diabodies, single domain antibody (e.g., a VH only antibody such as a nanobody), multispecific antibodies (e.g., bispecific antibodies) bifunctional antibodies, trifunctional antibodies, antibody-fusion proteins and any other modified configuration of the immunoglobulin molecule.
  • antigen-binding fragments thereof such as Fab, Fab', F(ab')2, Fv
  • scFv single-chain antibody
  • fusion proteins comprising an antibody portion (e.g., chimeric antigen receptor or CAR), humanized antibodies, chimeric antibodies, diabodies, single domain antibody (e.g., a
  • amino acid sequences at least about 80% identical to these regions can be incorporated into antibodies to obtain highly selective epitope-binding agents of choice.
  • these agents can be any one of e.g., polyclonal antibodies, monoclonal antibodies, synthetic antibodies, recombinant antibodies, antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, Fv), single-chain antibody (scFv), fusion proteins comprising an antibody portion (e.g., chimeric antigen receptor or CAR), humanized antibodies, chimeric antibodies, diabodies, single domain antibody (e.g., a VH only antibody such as a nanobody), multispecific antibodies (e.g., bispecific antibodies) bifunctional antibodies, trifunctional antibodies, antibody-fusion proteins and any other modified configuration of the immunoglobulin molecule that comprises an epitope binding region of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies, antibodies conjugated
  • the current disclosure also encompasses epitope-binding agents or antibodies disclosed herein conjugated to a detector molecule (labels, dyes, assay molecules) for example fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles and/or ligands, such as biotin fluorescent dyes, electrochemiluminescense dyes, metal-chelate complexes or labels.
  • a detector molecule labels, dyes, assay molecules
  • fluorescent labels for example fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles and/or ligands, such as biotin fluorescent dyes, electrochemiluminescense dyes, metal-chelate complexes or labels.
  • Fluorescent labels or fluorophores include rare earth chelates (europium chelates), fluorescein type labels including FITC, 5-carboxyfluorescein, 6-carboxy fluorescein; rhodamine type labels including TAMRA; dansyl; Lissamine; cyanines; phycoerythrins; Texas Red; and analogs thereof.
  • Fluorescent labels also include reactive and conjugated probes, e.g., Aminocoumarin, Fluorescein and Texas red, Alexa Fluor dyes, Cy dyes and DyLight dyes.
  • the fluorescent labels can be conjugated to an aldehyde group comprised in target molecule using the techniques disclosed herein.
  • Fluorescent dyes and fluorescent label reagents include those which are commercially available from Invitrogen/Molecular Probes (Eugene, Oreg., USA) and Pierce Biotechnology, Inc. (Rockford, III.).
  • Luminescent dyes or labels can be further subcategorized into chemiluminescent and electro-chemiluminescent dyes.
  • the different classes of chemiluminogenic labels include luminol, acridinium compounds, coe-lenterazine and analogues, dioxetanes, systems based on peroxyoxalic acid and their deriva-tives.
  • chemiluminogenic labels include luminol, acridinium compounds, coe-lenterazine and analogues, dioxetanes, systems based on peroxyoxalic acid and their deriva-tives.
  • acridinium based labels are used (a detailed overview is given in Dodeigne C. et al., Taianta 51 (2000) 415-439).
  • Electrochemiluminescense proved to be very useful in analytical applications as a highly sensitive and selective method. It combines analytical advantages of chemiluminescent analysis (absence of background optical signal) with ease of reaction control by applying electrode potential.
  • Ruthenium complexes especially [Ru (Bpy) 3 ] 2+ (which releases a photon at ⁇ 620 nm) regenerating with TPA (Tripropylamine) in liquid phase or liquid-solid interface are used as ECL-labels.
  • Electrochemiluminescent (ECL) assays provide a sensitive and precise measurement of the presence and concentration of an analyte of interest. Such techniques use labels or other reactants that can be induced to luminesce when electrochemically oxidized or reduced in an appropriate chemical environment. Such electrochemiluminescense is triggered by a voltage imposed on a working electrode at a particular time and in a particular manner. The light produced by the label is measured and indicates the presence or quantity of the analyte. Recently also Iridium-based ECL-labels have been described.
  • the directly detectable label/molecule is a chemiluminescent or an electrochemiluminescent label.
  • the light produced by the label is measured and directly or indirectly indicates the presence or quantity of the analyte.
  • Radioactive labels make use of radioisotopes (radionuclides), such as Iodine ( 125 l, 121 l, 124 l, 131 l), Carbon ( 14 C, 11 C), Sulfur ( 35 S), Tritium ( 3 H), Indium ( 121 ln), Flourine( 18 F), Phosphorus ( 32 P), Copper ( 64 Cu), Gallium ( 68 Gn), Yittrium ( 86 Y), Zirconium ( 89 Zr), Technetium ( 99 TC), Indum ( 111 ln), Xenon ( 133 Xe), Lutetium ( 177 Lu), or Astatine( 211 At).
  • radioisotopes radioisotopes
  • radioisotopes such as Iodine ( 125 l, 121 l, 124 l, 131 l), Carbon ( 14 C, 11 C), Sulfur ( 35 S), Tritium ( 3 H), Indium ( 121 ln), Flourine
  • Metal-chelate complexes suitable as labels for imaging and therapeutic purposes are well-known in the art (US 2010/0111856; U.S. Pat. Nos. 5,342,606; 5,428,155; 5,316,757; 5,480,990; 5,462,725; 5,428,139; 5,385,893; 5,739,294; 5,750,660; 5,834,456; Hnatowich et al, J. Immunol. Methods 65 (1983) 147-157; Meares et al, Anal. Biochem. 142 (1984) 68-78; Mir-zadeh et al, Bioconjugate Chem. 1 (1990) 59- 65; Meares et al, J.
  • the current disclosure also encompasses IF-binding agent complex.
  • IF-binding agent complex or IF-antibody complex refers to a complex formed by contacting an epitope-binding agent disclosed herein with IF or fragments thereof.
  • the IF is bound to B12.
  • the B12 bound IF or fragments thereof are referred to herein as the bound-IF as against unbound-IF, which does not have a bound B12.
  • the complex may be detected and quantified by various methods provided herein.
  • the complex can also be purified by methods known in the art.
  • the current disclosure encompasses IF-binding agent complexes that are detectible and quantifiable to a high degree of selectivity, sensitivity and reproducibility using the methods provided herein.
  • the complex may further comprise a second antibody or detection antibody conjugated to an enzyme or detectible molecule.
  • one or more components of the complex may be conjugated to for example fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles and/or ligands, such as biotin fluorescent dyes, electrochemiluminescense dyes, metal-chelate complexes or labels.
  • the complex may be detectible by one or more methods provided herein including immunoassays, such as the enzyme linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), immunohistochemistry (IHC), immunoprecipitation, and Western blotting.
  • immunoassays such as the enzyme linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), immunohistochemistry (IHC), immunoprecipitation, and Western blotting.
  • the current disclosure also encompasses epitope-binding agents comprising polyclonal antibodies manufactured by the process of i) isolating non-denatured B12 saturated IF (bound-IF) ii) immunizing a non-human animal by administering the bound-IF, iii) isolating immunoglobulins from the serum of the animal.
  • the current disclosure also encompasses epitope-binding agents comprising polyclonal antibodies manufactured by the process of i) isolating IF, ii) renaturing IF if denatured in a non-dentauring liquid medium, iii) saturating the isolated IF with B12 to form bound-IF, iv) immunizing an non-human animal by administering bound- IF, iii) isolating immunoglobulins from the serum of the animal.
  • the IF may be purified from a native source, for example from human gastrointestinal fluids.
  • the IF may be purified from a recombinant source.
  • Non-limiting examples of recombinant sources include viruses (for example a baculoviral vector), algae, fungi for example yeast expression systems, plants, cell lines (for example a mammalian line like CHO or plant cell lines).
  • the recombinant IF may be expressed by transient expression in genetic modification of plants, for example Nicotiana tabacum, Nicotiana benthamiana, and/or Arabidopsis thaliana.
  • These recombinant sources are engineered to express IF, that can be purified using well known techniques in the art.
  • Purification of IF from recombinant or native sources can be conducted using standard techniques including for example protein precipitation or chromatographic techniques or both, some of which are exemplified herein.
  • the purification of IF is conducted under non-denaturing conditions using a non-denaturing liquid medium comprising buffering agents, salts, osmotic agents or combinations thereof to maintain a physiological environment.
  • the IF can be purified under denaturing conditions and then refolded using a non-denaturing liquid medium comprising buffering agents, salts, osmotic agents and combinations thereof, to maintain a physiological environment.
  • Compositions of non-denaturing suitable for protein purification are well known in the art.
  • the non-denaturing liquid medium comprises B12. In some aspects, the non-denaturing liquid medium comprises about 500 pg/mL to about 1000 pg/mL, or about 1000 pg/mL to about 1500 pg/mL, 1500 pg/mL to about 2000 pg/mL, 2000 pg/mL to about 2500 pg/mL, 2500 pg/mL to about 3000 pg/mL, 3000 pg/mL to about 3500 pg/mL, 3500 pg/mL to about 4000 pg/mL, 4000 pg/mL to about 4500 pg/mL, 4500 pg/mL to about 5000 pg/mL of B12.
  • epitope binding agents for example, polyclonal antibodies can be generated using suitable methods available in the art. Typically these methods involve immunizing a non-human animal by administering the polypeptide of interest, for example bound-IF.
  • the polypeptide of interest is typically administered into the non- human animal’s blood stream with a suitable adjuvant. Multiple administration of the polypeptide can be conducted to boost the immune system.
  • the blood of the animal can then be obtained, and separation of serum and cellular fraction may be conducted.
  • the serum fraction contains the polyclonal antibodies.
  • the polyclonal antibodies can be further purified, or the serum can be directly used.
  • further purification may be achieved by for example, one or more steps of antibody precipitation, one or more chromatographic purifications or combinations thereof.
  • high affinity antibodies can be obtained by further purifying a population of antibodies against a chromatographic column comprising bound-IF and then separating the bound-IF to obtain unbound antibodies.
  • the epitope binding agents can be used at a suitable concentration range as determined by a person skilled in the art as demonstrated in the examples herein.
  • the present disclosure encompasses methods to assay bodily fluid/s from a subject in need thereof, to detect or quantitate or both, IF or fragments thereof.
  • An important and novel aspect of the current disclosure is the ability to detect very low levels of IF or fragments thereof in biological fluids using the epitope binding agents, for example polyclonal antibodies as disclosed herein. These methods have been optimized for clinical use.
  • the optimization of certain methods as provided herein is responsible for the high accuracy and sensitivity of these assays.
  • the use of B12 in the buffer system used in these assays though not essential or limiting, greatly improves the sensitivity of these assays. This enables the use of the methods provided herein to easily, non-invasively, and with high degree of sensitivity and reproducibility determine IF-levels in a subject in need thereof to predict certain IF-related disease states.
  • the current disclosure encompasses methods of using the epitope-binding agent and antibodies provided herein to detect or quantify or both, IF or fragments thereof in a sample using novel methods provided herein or known to those of skill in the art, including immunoassays, such as the enzyme linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), immunohistochemistry (IHC), immunoprecipitation, and Western blotting.
  • immunoassays such as the enzyme linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), immunohistochemistry (IHC), immunoprecipitation, and Western blotting.
  • the epitope-binding agent for use in these methods may comprise one or more of naturally occurring antibodies, non-naturally occurring antibodies; polyclonal antibodies, monoclonal antibodies, synthetic antibodies, recombinant antibodies, antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, Fv), single-chain antibody (scFv), fusion proteins comprising an antibody fragment (e.g., chimeric antigen receptor or CAR), humanized antibodies, chimeric antibodies, diabodies, single domain antibody (e.g., a VH only antibody such as a nanobody), multispecific antibodies (e.g., bispecific antibodies) bifunctional antibodies, trifunctional antibodies, antibody-fusion proteins and any other modified configuration of the immunoglobulin molecule that comprises an epitope-binding region that binds the IF.
  • polyclonal antibodies monoclonal antibodies, synthetic antibodies, recombinant antibodies, antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, F
  • ELISA refers to an enzyme-linked immunosorbent assay which can quickly detect and quantify minute amounts (less than a nanogram) of antigen in a biological sample. Any suitable ELISA protocol known in the art can be used in the methods of the present disclosure. ELISA is characterized by a series of antibody interactions to detect the presence of a target in a sample in vitro. ELISAs can include a direct assay, an indirect assay, or a capture assay (i.e. , a sandwich assay). In a direct assay, an antigen is bound to a surface. A primary antibody conjugated to an enzyme is applied to the surface, and the primary antibody binds the antigen.
  • a substrate is then added that is acted on by the enzyme, producing a detectable signal, which indicates the presence of the antigen on the surface.
  • an antigen is bound to a surface.
  • a primary antibody is then applied to the surface, and the primary antibody binds the antigen.
  • a secondary antibody (detection antibody) conjugated to an enzyme is then applied to the surface. The secondary antibody can bind the primary antibody (capture antibody) or the antigen-antibody complex.
  • a substrate is then added that is acted on by the enzyme, producing a detectable signal, which indicates the presence of the antigen on the surface.
  • the primary antibody may be a capture antibody.
  • the primary antibody may be a detector antibody.
  • capture assay In a “capture assay” or “sandwich ELISA”, one or more capture antibody is bound to an inert polymer support, such as a plastic tray with molded wells or a strip and then exposed to the biological sample. Unbound proteins are washed away and a detection antibody that reacts with the antigen at a different epitope than the test antibody reacts with is added.
  • This second antibody has an enzyme attached to it that converts a colorless or nonfluorescent substrate into a colored or fluorescent product.
  • the amount of second antibody bound, and hence the amount of protein antigen present in the original biological sample, is determined by the quantification of the intensity of color or fluorescence produced, (see Instant Notes: BioChemistry, 2nd edition, B. D. Hames and N. M. Hooper, Springer-Verlag New York 2000, pages 112-114 for an introduction to the general principles of ELISA assays).
  • a “capture antibody,” as used herein, refers to an antibody that specifically bind a target molecule, e.g., a form of IF, in a sample.
  • a capture antibody may be attached to a solid support surface, such as, for example but not limited to, a plate or a bead, e.g., a paramagnetic bead.
  • the capture antibody may bind to an antigen immobilized on a plate or a bead as in a capture or sandwich ELISA assay.
  • the capture antibody forms a complex with the target molecule such that the antibody-target molecule complex (for example an IF- binding agent complex) can be separated from the rest of the sample.
  • such separation may include washing away substances or material in the sample that did not bind the capture antibody.
  • a “detection antibody,” or “detecting antibody” as used herein, refers to an antibody that specifically binds a target molecule in a sample or in a sample-capture antibody combination material for example an IF-binding agent complex. Under certain conditions, the detection antibody forms a complex with the target molecule or with a target molecule-capture antibody complex.
  • a detection antibody is capable of being detected either directly through a label, which may be amplified, or indirectly, e.g., through use of another antibody that is labeled and that binds the detection antibody.
  • the detection antibody is typically conjugated to a moiety that is detectable by some means, for example, including but not limited to, biotin, HRP, fluorescent molecules, radioactive isotopes etc.
  • the capture antibody is typically conjugated to a moiety that is detectable by some means, for example, including but not limited to, biotin, HRP, fluorescent molecules, radioactive isotopes.
  • the detecting antibody may be conjugated to a moiety detectible by some means for example enzyme, for example, biotin, horse radish peroxidase, fluorescent molecules, radioactive isotopes, alkaline phosphatase, to aid in the detection of the complex.
  • a secondary antibody conjugated to an enzyme e.g., alkaline phosphatase
  • an enzyme e.g., alkaline phosphatase
  • the complex will then be detected using a luminescent substrate, for example, Ultra LITETM (NAG Research Laboratories); SensoLyte® (AnaSpec); SuperSignal ELISA Femto Maximum Sensitivity Substrate (Thermo Scientific); SuperSignal ELISA Pico Chemiluminescent Substrate (Thermo Scientific); CPSD (disodium 3-(4-methoxyspiro ⁇ 1 ,2-dioxetane-3,2'-(5'- chloro)tricyclo[3.3.1 ,13,7]decan ⁇ -4-yl)phenyl phosphate; Tropix, Inc).
  • a luminescent substrate for example, Ultra LITETM (NAG Research Laboratories); SensoLyte® (AnaSpec); SuperSignal ELISA Femto Maximum Sensitivity Substrate (Thermo Scientific); SuperSignal ELISA Pico Chemiluminescent Substrate (The
  • the sandwich ELISA provided herein comprises the use of a capturing antibody comprising at least one of rabbit F2832 y- globulin 0994 or a monoclonal mouse anti human IF antibody and a detection antibody comprising a biotinylated rabbit F2831 y-globulin 0994.
  • the ELISA is a “chemiluminescent ELISA.”
  • a “chemiluminescent ELISA” or a “luminescent assay” or a “chemiluminescent assay” is a type of ELISA that uses the emission of light as an indicator of the presence of a target.
  • the intensity of the emitted light can then be measured and correlated with the abundance of the target in a sample.
  • an enzyme converts a substrate to a reaction product that emits photons of light.
  • Luminescence is described as the emission of light from a substance as it returns from an electronically excited state to a ground state.
  • Chemiluminescence is light produced by a chemical reaction. When the excited intermediates return to their stable ground state, a photon is released, which is detected by a sensor in a luminescent signal instrument.
  • the intensity of the luminescent signal can be expressed in chemiluminescence units, wherein the greater the number of detected chemiluminescence units is, the higher the titer of the target in the biological sample.
  • the chemiluminescent enzyme comprises alkaline phosphatase (AP). In some embodiments, the chemiluminescent enzyme comprises horse radish peroxidase (HRP). In some embodiments, the chemiluminescent enzyme comprises beta galactosidase and beta lactamase.
  • AP alkaline phosphatase
  • HRP horse radish peroxidase
  • beta galactosidase and beta lactamase any chemiluminescent enzyme and substrate can be used in the methods disclosed herein.
  • Suitable antibody detection molecules include enzyme labels, such as, horse-radish peroxidase (HRP) and glucose oxidase; radioisotopes, such as iodine ( 125 l, 121 1, 124 l, 131 1), Carbon ( 14 C, 11 C), Sulfur ( 35 S), Tritium ( 3 H), Indium ( 121 ln), Flourine( 18 F), Phosphorus ( 32 P), Copper ( 64 Cu), Gallium ( 68 Gn), Yittrium ( 86 Y), Zirconium ( 89 Zr), Technetium ( 99 TC), Indum ( 111 ln), Xenon ( 133 Xe), Lutetium ( 177 Lu), Astatine( 211 At), or haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored
  • an enzyme an enzyme tag
  • a colored product upon contact with a chromogenic substrate.
  • suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and/or glucose oxidase.
  • Such detection molecules can be used to label an epitope-binding agent, antibody or antigen-binding fragment, or variants thereof described herein.
  • a second antibody or detection antibody or antigen-binding fragment thereof that recognizes a different epitope on IF or recognizes an anti-IF antibody or antigen-binding fragment or variant thereof described herein can be labeled and used in combination with an anti-IF antibody or antigen-binding fragment thereof to detect IF protein levels.
  • such a secondary antibody, or detection antibody or antigen-binding fragment thereof e.g., an anti-human or anti-rodent antibody
  • an enzyme e.g., horseradish peroxidase
  • a substrate of the enzyme e.g., 3,3'- diaminobenzidine (DAB)
  • attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid an-hydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetra-chloro-3a- 6a-diphenylglycouril-3 attached to the antibody (U.S. Pat. Nos. 4,472,509 and 4,938,948, each incorporated herein by reference).
  • DTPA diethylenetriaminepentaacetic acid an-hydride
  • ethylenetriaminetetraacetic acid N-chloro-p-toluenesulfonamide
  • tetra-chloro-3a- 6a-diphenylglycouril-3 attached to the antibody
  • Antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate.
  • a coupling agent such as glutaraldehyde or periodate.
  • Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate.
  • Assaying for the expression level of IF protein is intended to include qualitatively or quantitatively measuring or estimating the level of IF protein or variants or fragments thereof in a first sample either directly (e.g., by determining or estimating absolute protein level) or relatively (e.g., by comparing to IF protein level in a second sample or standard).
  • IF levels in the sample can be measured or estimated and compared to a standard IF protein level, the standard being determined from a second biological sample that is not diseased or being determined by averaging levels from a population of samples that are not diseased.
  • the “standard” IF polypeptide level is known, it can be used repeatedly as a standard for comparison.
  • Samples as used herein may vary depending on the application. For example, it may be a biological sample or a non-biological sample.
  • the term non- biological sample may include synthetic peptides, buffers, non-clinical fluids, surfaces etc.
  • biological sample includes any biological specimen obtained from an individual. Suitable samples for use in the present invention include, without limitation, whole blood, plasma, serum, saliva, urine, stool (i.e., feces), tears, amniotic fluid and any other bodily fluid, or a tissue sample (i.e., biopsy) such as a small intestine or colon sample, and cellular extracts thereof (e.g., red blood cellular extract).
  • the sample is a fluid sample like blood, plasma, serum, urine or gastric sample.
  • the sample is a serum sample.
  • samples such as serum, plasma, saliva, amniotic fluid and urine are well known in the art (see, e.g., Hashida et al., J. Clin. Lab. Anal., 11 :267-86 (1997)).
  • samples such as serum samples can be diluted prior to the analysis of marker levels.
  • the sample may be a fluid sample, a solid sample or a sample bound to a solid surface like matrices, beads, strips, solid substrate material or membrane (e.g., plastic, nylon, paper), plates etc.
  • the samples may be concentrated or diluted such that the IF levels are within a preferred range based on the application and detection method.
  • buffers used in the assays may include for example phosphate buffer, phosphate buffer saline (PBS), PBST, Tris buffer, TBS, TBST, etc.
  • these buffers may further comprise B12.
  • B12 is needed to form red blood cells and DNA. It is also a key player in the function and development of brain and nerve cells. IF binds B12 and undergoes a conformation change from it’s unbound- to bound-form.
  • the amount of B12 added to the assays may vary depending on applications.
  • the assay buffer may comprise about 1 nM of B12 in 0.1 % PBS. In some aspects, the assay buffer may comprise about 0.1 nM to about 10 nM B12.
  • the assay buffer may comprise 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1 nM, 1.1 nM, 1.2 nM, 1.3 nM, 1.4 nM, 1.5 nM, 1.6 nM, 1.7 nM, 1.8 nM, 1.9 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9nM or 10 nM or any intermediate amounts of B12 suitable for the application.
  • the methods provided herein may comprise further steps including but not limited to detection methods, methods for calibration, detection, data interpretation, storage.
  • the colorimetric assay as provided herein can be detected and optionally quantified and analyzed using an image capture device such as a digital camera or a desktop scanner attached to a computer.
  • image capture device such as a digital camera or a desktop scanner attached to a computer.
  • concentration values of known standard elements can be used to generate standard curves.
  • Concentration values for unknown analytes can be analyzed using the standard curve for each analyte to calculate actual concentrations. Values for each analyte can be identified based on the spotting position of each capture element within the array.
  • sensitivity refers to the lowest value of analyte (for example IF and/or fragments thereof) that the assay can statistically differentiate from background.
  • the epitope-binding agents and antibodies provided herein provide a lower limit of detection defined as the concentrations corresponding to a signal, 2 standard deviations (SD) above the IF-free calibrator of about 0.01 pmol/L to about 10 nmol/ L depending on the application and specific assay attributes.
  • SD standard deviations
  • the sensitivity of the assay is in the pmol/ L range.
  • the sensitivity is in the range of about 0.1 pmol/L to about 0.5 pmol/ L, or about 0.5 pmol/L to about 1 pmol/ L, or about 1 pmol/L to about 5 pmol/ L, or about 5 pmol/L to about 10 pmol/ L, or about 10 pmol/L to about 15 pmol/ L, or about 15 pmol/L to about 20 pmol/ L, or about 20 pmol/L to about 25 pmol/ L, or about 25 pmol/L to about 30 pmol/ L, or about 30 pmol/L to about 35 pmol/ L, or about 35 pmol/L to about 40 pmol/ L, or about 45 pmol/L to about 50 pmol/ L, or about 50pmol/L to about 100pmol/L, or about 100 pmol/L to about 500 pmol/L.
  • the sensitivity of the assay is such that it allows for a measurement range of 0.01-1000 pmol/L.
  • the assay system provided herein can detect less than 0.1 , or less than 0.2, or less than 0.3, or less than 0.4, or less than 0.5, or less than 0.6, or less than 0.7, or less than 0.8, or less than 0.9, or less than 1 , or less than 2, or less than 5, or less than 10, or less than 20, or less than 50, or less than 100 pmol/ul, or less than 500 pmol/L
  • these assays are also optimized to be effective for clinical use. Assay performance can be analyzed in a number of different ways.
  • the mean background for the assay was about OD: ⁇ 0.02.
  • the defined lowest detectable value of 0.3 pmol/L had an OD of -0.04, that is OD 0.02 higher than the background in the examples provided herein. This results in a high signal to noise ratio making it possible to successfully detect pmol/L quantities of IF, the level present in blood and other body fluids.
  • the present disclosure encompasses clinical and non- clinical use of the compositions, methods, and assays provided herein to detect or quantify IF or both.
  • a key aspect of the current disclosure is the ability to detect miniscule levels (picomolar) of IF or fragments or combination thereof in bodily fluid/s from a subject in need thereof.
  • “Gastric IF” or “IF” or “B12-binding IF” is a glycoprotein produced by the parietal cells of the stomach in humans. It is necessary for the absorption of B12 in the distal ileum of the small intestine. IF is secreted by the stomach, and so is present in the gastricjuice as well as in the gastric mucous membrane.
  • Changes in gastric IF levels is linked to multiple IF-related disorders including but not restricted to pernicious anemia, megaloblastic anemia, gastritis, atrophic gastritis, peptic ulcers, gastrinoma, gastric tumors, gastric ulcers caused by infections like those caused by H. pylori, genetic disorders linked to mutations in GIF gene, damages to stomach lining or portions of stomach parietal cells, damages due to bariatric surgery, gastric bypass surgery, gastric problems linked to certain neurological conditions, and certain autoimmune conditions like presence of anti-IF antibodies or anti-stomach parietal cell antibodies.
  • these disease states or conditions may result in reduced (to none) levels of IF or increased levels of IF (as in some tumors) in a subject.
  • the disclosure encompasses use of the compositions and methods provided herein to detect IF-levels in a subject in need thereof.
  • the detection of levels of IF can be important in the diagnosis of diseases and conditions provided above.
  • Commercially available antibodies and methods to detect IF have low sensitivities and can detect IF in gastric fluids, where it is present in nmol/L concentrations.
  • gastric fluids can only be obtained using invasive procedures and the process is timeconsuming and complex.
  • the methods presented herein can detect IF in serum and urine samples with IF levels in the pmol/L range.
  • the methods presented herein are able to detect pmol/L range of IF due to the use of high sensitive antibodies made using the methods disclosed herein.
  • these antibodies are raised against bound-IF as provided herein.
  • these high sensitive antibodies can be used in highly optimized clinical and non-clinical assay systems as disclosed herein. This allows for the use of these methods for quick and reliable detection for prevention, diagnosis, treatment, and testing of treatment efficacy in subjects.
  • IF polypeptide levels in the sample can be measured or estimated and compared to a standard and/or threshold IF level, the standard being determined from a second biological sample that is not diseased or being determined by averaging levels from a population of samples that are not diseased.
  • IF polypeptide levels in the sample can be measured or estimated and compared to a standard and/or threshold IF level, the standard being determined from a second biological sample that is not diseased or being determined by averaging levels from a population of samples that are not diseased.
  • the patient’s own previously determined levels can be used as a standard, for instance the level of a preoperative patient can be used as a standard for comparison of post-operative results. All these methods can be used to determine threshold levels to determine disease conditions.
  • the threshold may vary depending on the patient population, the disease conditions and the fluid sample used.
  • the subject may be a mammal. In some aspects, the subject is human. In some aspects, the subject is suspected of having, or has, one or more of an IF-related condition like pernicious anemia, megaloblastic anemia, gastritis, atrophic gastritis, peptic ulcers, gastrinoma, gastric tumors, gastric ulcers caused by infections like those caused by H. pylori, genetic disorders linked to mutations in GIF gene, damages to stomach lining or portions of stomach parietal cells, damages due to bariatric surgery, gastric bypass surgery, gastric problems related to certain neurological conditions, and certain autoimmune conditions like presence of anti-IF antibodies or antistomach parietal cell antibodies.
  • an IF-related condition like pernicious anemia, megaloblastic anemia, gastritis, atrophic gastritis, peptic ulcers, gastrinoma, gastric tumors, gastric ulcers caused by infections like those caused by H. pylori, genetic disorders linked to
  • the subject is not capable of, or has diminished capacity to absorb B12.
  • the subject may be pre-operative or postoperative to a procedure including, but not restricted to, gastric bypass surgery, greater fundus removal, bariatric surgery, ileostomy procedure etc.
  • the subject may have sustained injury that results in low secretion of IF.
  • the subject may have none to reduced levels of IF.
  • the subject may have enhanced levels of IF for instance in some gastrinomas.
  • the patient may be in need of evaluation to determine the efficacy of administering B12.
  • the subject may be a newborn, less than 6 months old, 6 months or older, 12 months or older, 18 months or older, 2 years or older, 4 years or older, 6 years or older, 10 years or older, 13 years or older, 16 years or older, 18 years or older, 21 years or older, 25 years or older, 30 years or older, 35 years or older, 40 years or older, 45 years or older, 50 years or older, 60 years or older, 65 years or older, 70 years or older, 75 years or older, 80 years or older, 85 years or older, 90 years or older, or 1 -12 months or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16 ,18, 20, 21 , 24, 25, 27, 28, 30, 33, 35, 37, 39, 40, 42, 44, 45, 48, 50, 52, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100, 101 , 102, 103
  • compositions and methods provided herein may also be used in non-clinical, for example laboratory settings.
  • the compositions and methods provided herein may be used for immunoassays ranging from immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), immunohistochemistry (IHC), immunoprecipitation, purification, and Western blotting on laboratory samples.
  • ELISA immunosorbent assay
  • FACS fluorescence-activated cell sorting
  • IHC immunohistochemistry
  • purification purification
  • Western blotting Western blotting
  • kits comprising at least one epitope-binding agent or a composition comprising the epitope-binding agent described herein.
  • the kit comprises an epitope-binding agent or antibody made using the methods as disclosed herein.
  • such a kit is a packaged combination including the basic elements of: a capture antibody comprised of an anti-IF antibody; a detectable (labeled or unlabeled) antibody that binds to the antibody of interest or another epitope on IF; and instructions on how to perform the assay method using these reagents. These basic elements are defined hereinabove.
  • the kit may further comprise a solid support for the capture reagents, which may be provided as a separate element or on which the capture reagents are already immobilized.
  • the capture antibody may already be immobilized on the solid support for example a plate, matrix, paper strip, plastic strip, or beads.
  • the capture antibodies in the kit may be immobilized on a solid support, or they may be immobilized on such support that is included with the kit or provided separately from the kit.
  • the capture reagents are coated on or attached to a solid material (for example, a microtiter plate, beads or a comb).
  • the detectable antibodies may be labeled antibodies detected directly or unlabeled antibodies that are detected by labeled antibodies directed against the unlabeled antibodies raised in a different species or targeted to another epitope of IF.
  • the kit will ordinarily include substrates and cofactors required by the enzyme; where the label is a fluorophore, a dye precursor that provides the detectable chromophore; and where the label is biotin, an avidin such as avidin, streptavidin, or streptavidin conjugated to HRP or [3-galactosidase with MUG.
  • the kit also typically contains the analyte for example native, recombinant or synthetic human IF and/or fragments thereof of interest as a standard as well as other additives such as stabilizers, washing and incubation buffers, and the like.
  • analyte for example native, recombinant or synthetic human IF and/or fragments thereof of interest as a standard as well as other additives such as stabilizers, washing and incubation buffers, and the like.
  • the components of the kit will be provided in predetermined ratios, with the relative amounts of the various reagents suitably varied to provide for concentrations in solution of the reagents that substantially maximize the sensitivity of the assay.
  • the reagents may be provided as dry powders, usually lyophilized, including excipients, which on dissolution will provide for a reagent solution having the appropriate concentration for combining with the sample to be tested.
  • kits comprising an epitope-binding agent for example an antibody as described herein is for use in a method as described herein (e.g., in a method of detecting IF and/or fragments thereof).
  • the kit further comprises an anti-IF antibody coated or attached to a comb for use in a method of detecting or quantifying IF in biological fluids.
  • the kit may comprise a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, assay plates, strips, matrices etc.
  • the containers may be formed from a variety of materials such as glass, plastic, paper etc.
  • the kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • a “package insert” is used to refer to instructions customarily included in commercial packages of diagnostic products, that contain information about usage etc.
  • kits may be affixed to packaging material or may be included as a package insert. While the instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” may include the address of an internet site that provides the instructions.
  • the term “corresponding” as used generally with reference to antibody-antigen binding complexes for example, an antibody corresponding to an antigen will bind to the antigen under physiologic conditions.
  • the bound antibody-antigen is referred to as an antibody-antigen binding complex.
  • derivative when used herein with reference to a polypeptide, refers to a polypeptide related to a wild type polypeptide, for example either by amino acid sequence, structure (e.g., secondary and/or tertiary), activity (e.g., enzymatic activity) and/or function.
  • Derivatives, variants and fragments of a polypeptide can comprise one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof compared to a wildtype polypeptide.
  • a part or fragment of a polypeptide may correspond to at least 1 %, at least 2%, at least 3 %, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40% of the length of a polypeptide, such as a polypeptide having an amino acid sequence having at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 99% of the length (in amino acids) of the polypeptide.
  • Human IF was purified from human gastric juice such that the protein is saturated with B12. The purification was done without a protein denaturation step in order to maintain close to native structural conformation.
  • Two polyclonal rabbit anti-human IF antibodies were custom-made against the pure human IF by immunization of two rabbits (F2831 and F2832) (DAKO A/S) followed by purification of the y-globul in fraction of the rabbit serum.
  • the freeze-dried rabbit F2831 y-globulin 0994 was biotinylated by coupling active biotin to the antibody and inactivating excess biotin with lysine and used for the detecting antibody.
  • Recombinant human IF was used as calibrator by dissolving 20 nmol/ml IF (Cobento Biotech A/S (now Xeragenx LLC)) in 0.1 % phosphate buffered albumin (PBA) made by dissolving bovine serum albumin (Sigma-Aldrich, cat. no. A7030) in a 0.1 M phosphate buffer (VWR International, cat. no. AMPQ40654), pH 8.
  • the calibrators were prepared to cover a concentration range of 0.8 to 50 pmol/L by dilution in assay buffer (assay buffer details are given below), which was also used as a zero calibrator. All calibrators were stored a -20°C until used.
  • a low control IF concentration of 4 pmol/L was made from a pool of anonymous donor serum that was centrifuged for 9 minutes at 1850 x g at room temperature.
  • a high control IF concentration of 30 pmol/L was made by spiking 39.8 ml of the same serum pool with 122 pl de-pepsinated gastric juice.
  • the gastric juice was collected by medical staff as part of a diagnostic test for acidity (Bispebjerg Hospital, Copenhagen, Denmark). Excess samples of gastric juice - used in this study - were collected with no information allowing the samples to be traced back the donor.
  • the two controls were aliquoted and stored at -20°C until usage. When running the ELISA, the high and low controls were placed both before and after the samples on the microtiter plate.
  • Prototype ELISA ELISA microtiter plates (F96 Maxisorp 442404, Nunc) were coated with 1-pg anti-IF catching (rabbit F2832 y-globulin 0994) antibody. The coated plates were stored at -20°C and removed to room temperature one hour before usage. The plates were washed three times in 350 pl washing buffer (VWR International, cat. no. AMPQ15265) containing 1.5 mM NaH2PO4, 8.5 mM Na2HPO4, 145 mM NaCI, 1 g/L Tween 20, and demineralized H2O, pH 7.4, by the Biotec ELx50 microtiter plate washer (Holm & Halby A/S).
  • 50-pl assay buffer (1 nM final B12 in 0.1 % PBS) was added to each well. Subsequently, 50 pl of sample or 50 pl control was added to the wells. After and O/N incubation at 4°C; with gentle shaking, the plates were washed three times in washing buffer.
  • Detection antibody comprising the biotinylated rabbit F2831 y-globulin 0994 polyclonal antibody was diluted 1 :500 to a concentration of 0.868 mg/ml in 0.1 % PBA (without B12) and 100 pl of this solution was added to each well, 0.174 pg/well, and incubated and incubated for 3 hours at room temperature with gentle shaking.
  • This reagent contained 6 pl of avidinperoxidase-conjugate (Sigma, cat. no. A7419) diluted 1 :30, 120 pl lysozymes, and 12 ml POD buffer (VWR International, cat. no. AMPQ42067) pH 7.4 made from 1.5 mM NaH2PO4, 8.5 mM Na2HPO4, and 400 mM NaCI in demineralized water.
  • the plates were incubated for 30 min with the POD reagent at room temperature with gentle shaking.
  • the plates were then washed three times in washing buffer before addition of color reagent; 100 pl TMB ONE Ready-to-use Substrate (KEM EN TEC, cat. no. 4380A) to each well.
  • the plates were incubated for 13-14 minutes at room temperature with gentle shaking before the color reaction was stopped by adding of 100-pl of 0.1 M phosphoric acid made by diluting 136.3 ml of 85% orthophosphoric acid (VWR International, cat. no. 20621 ) in 2 L ELGA water.
  • VWR International cat. no. 20621
  • the color development was measured at 450 nm and corrected for absorbance at 620 nm on the MultiSkan Ascent (Thermo).
  • Sandwich ELISA ELISA microtiter plates (F96 Maxisorp 442404, Nunc) were coated with 1 -pg anti-IF catching (capture) antibody. The coated plates were stored at -20°C and removed to room temperature one hour before usage. The plates were washed three times in 350 pl washing buffer (VWR International, cat. no. AMPQ15265) containing 1.5 mM NaH2PO4, 8.5 mM N32HPO4, 145 mM NaCI, 1 g/L Tween 20, and demineralized H2O, pH 7.4, by the Biotec ELx50 microtiter plate washer (Holm & Halby A/S). Then, 50-pl assay buffer was added to each well.
  • VWR International cat. no. AMPQ15265
  • the B12 assay buffer was prepared by adding B12 (cyanocobalamin, Sigma-Aldrich, cat. no. V2876) to 0.1 % PBA to a final concentration of 1 nM B12. Addition of B12 to the buffer ensured that all IF was saturated with B12 before analysis and greatly improved the sensitivity of the assay as provided in FIG. 1. Next, 50 pl sample or 50 pl control was added to the wells. Calibrators were prepared by dilution in the assay buffer (see details above), and 100 pl calibrators were added directly to the wells (without pre-addition of 50 pl assay buffer).
  • the detection antibody biotinylated rabbit F2831 y-globulin 0994 polyclonal antibody
  • the detection antibody was diluted 1 :500 to a concentration of 0.868 mg/ml in 0.1 % PBA (without B12) and 100 pl of this solution was added to each well, 0.174 pg/well, and incubated 2 hours at room temperature with gentle shaking.
  • the plates were then washed three times again in washing buffer before addition of 100 pl of horseradish peroxidaseavidin to each well.
  • This reagent contained 6 pl of avidin-peroxidase-conjugate (Sigma, cat. no.
  • the plates were incubated for 13- 14 minutes at room temperature with gentle shaking before the color reaction was stopped by adding of 100-pl of 0.1 M phosphoric acid made by diluting 136.3 ml of 85% orthophosphoric acid (VWR International, cat. no. 20621 ) in 2 L ELGA water.
  • VWR International cat. no. 20621
  • the color development was measured at 450 nm and corrected for absorbance at 620 nm on the MultiSkan Ascent (Thermo).
  • the calibration curve was computed by plotting the absorbance of the calibrators and constructing a cubic spline curve with linear scale on both axes. The results for samples and controls were read from this curve.
  • the final IF ELISA assay was validated by the following methods: to study linearity, intra-assay imprecisions and recovery, two serum stocks with high (26 pmol/L) and low (3.3 pmol/L) concentration of IF were pooled to give the expected IF concentrations of 26, 21 , 17, 12, 7.8, and 3.3 pmol/L.
  • the intra-assay imprecision (CV% intra) (variation within the same microtitre plate) was calculated for each pool in each of the six runs by finding the standard deviation of the quadruple results, dividing that by the quadruple mean, and multiplying by 100.
  • the mean intra-assay imprecision for each pool was determined by taking the average of the six individual CVs (one determined for each run), and the overall intra-assay variation was determined by taking the average of these calculated CVs (one determined for each pool based on all runs).
  • Recovery was determined on each of the 20 serum pools with mean IF concentration of 14 pmol/L made from pooling of 40% of the low control and 60% of the high control. Recovery was defined as: (final concentration - initial concentration (low control)) I added concentration (high control). The overall recovery was calculated by taking the average of the recovery of the 20 serum pools.
  • Non-specific binding, sensitivity and background Recombinant IF was employed as calibrator, and a standard calibration curve was made as depicted in FIG. 3.
  • the lower limit of detection (sensitivity) defined as the concentrations corresponding to a signal 2 SD above the IF-free calibrator, was 0.3 pmol/L, giving a sensitive assay with a measurement range of 0.3-50 pmol/L.
  • Mean background for the assay was about OD: ⁇ 0.02.
  • the defined lowest detectable value of 0.3 pmol/L had an OD of ⁇ 0.04, that is OD 0.02 higher than the background. This results in a high signal to noise ratio making it possible to successfully detect pmol/L quantities of IF, the level present in blood and other body fluids.
  • the intra-assay imprecision (CV%intra) was calculated at six different concentrations and ranged from 4.8% to 8.4% with a mean intra-assay imprecision of 6.9% (FIG 3).
  • the total imprecision (CV%total) was found to be 16.8% at 4 pmol/L and 13.3% at 30 pmol/L based on 65 independent measures performed over 3 years with a mean total imprecision of 15%.
  • sample stability and effect on test results When testing sample stability, the serum IF concentration varied between 1.6% and 8.5% for the four serum samples (serum IF from 5 - 10 pmol/L) through eight freeze-thaw cycles over a period of 15 weeks. No systematic decline or increase of the concentration was seen (data not shown). After 33 months, the four serum samples were reanalyzed for evaluation of stability after longterm storage. IF concentration was found to be between 12% and 16% higher than baseline, which is within common acceptable criteria ( ⁇ 20%) for stability data on ligand binding assays.
  • the established ELISA allowed for quantification of IF down to 0.3 pmol/L with an overall intra-assay imprecision of 6.9% and a total imprecision of 15%. Notably, no alternations of IF occurred upon repeat freeze-thawing cycles, nor upon storage of samples for up to 33 months. Thus, the assay may prove very useful also for analyzing archival serum samples.
  • the assay is highly specific for IF as judged by size exclusion chromatography and the lack of cross reactivity with the B12-binding proteins, HC and TC, that are structurally related to IF and occur in nanomolar concentrations in serum.
  • the samples were immediately anonymized upon collection, and centrifuged (10 min. at 2300 x g) within four hours. Serum was stored at -20°C for later analysis of IF as described above for the final ELISA assay. No ethical approval was necessary according to national law.
  • Sandwich ELISA Assay provided in Example 1 was used to determine interindividual (SDinter) and intraindividual (SDday-to-day and SDwithin-day) variations for serum IF based on the remaining cohort of 20 healthy individuals.
  • SDinter was determined on the 9 o'clock Day 1 measures (one measure per individual).
  • the SDday-to-day was determined by finding the SD on the three 9 o'clock measures (Day 1 , 2, and 3) from the same individual and calculating the total mean SD of the 20 individuals.
  • the SDwithin-day was determined by finding the SD on all Day 2 measures (at 9, 12, 15, 18, and 21 o'clock) from the same person and calculating the total mean SD of the 20 individuals.
  • Urine samples were collected from one male and one female. The samples were dialyzed, lyophilized, and redissolved so that the final concentration factor for the original sample was ⁇ 40-fold. For comparison blood samples were collected from the same individuals. The IF concentration was measured using the sandwich ELISA assay as provided in Example 1. Results are provided in Table 2 below.

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Abstract

La présente invention concerne des compositions et des dosages pour détecter l'IF gastrique dans des fluides corporels tels que le sang et l'urine, et l'utilisation de telles compositions et dosages dans la détection de maladies associées à IF.
PCT/US2023/024980 2022-06-10 2023-06-09 Détection du facteur intrinsèque (if) gastrique dans des échantillons de fluide WO2023239935A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2110019C (fr) * 1993-05-28 2006-01-24 Karel Newman Immunodetection de la vitamine b12, reactifs et trousses
GB2461076A (en) * 2008-06-20 2009-12-23 James Love Devices and methods for the separation of blood serum from a blood sample
WO2020236903A1 (fr) * 2019-05-20 2020-11-26 Syracuse University Formulations pharmaceutiques et procédés d'administration d'un agent thérapeutique, de diagnostic ou d'imagerie à cd206

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2110019C (fr) * 1993-05-28 2006-01-24 Karel Newman Immunodetection de la vitamine b12, reactifs et trousses
GB2461076A (en) * 2008-06-20 2009-12-23 James Love Devices and methods for the separation of blood serum from a blood sample
WO2020236903A1 (fr) * 2019-05-20 2020-11-26 Syracuse University Formulations pharmaceutiques et procédés d'administration d'un agent thérapeutique, de diagnostic ou d'imagerie à cd206

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* Cited by examiner, † Cited by third party
Title
"Enzyme-linked Immunosorbent Assay Kit For Gastric Intrinsic Factor (GIF", INSTRUCTION MANUAL, July 2013 (2013-07-01), pages 1 - 8, Retrieved from the Internet <URL:file://core-fs-01/FTEHome$/ks03/Documents/US23_24980/NPL_MyBioSource.pdf> [retrieved on 20230906] *
ANDRÈS E., SERRAJ K., ZHU J., VERMORKEN A.J.M.: "The pathophysiology of elevated vitamin B12 in clinical practice", INTERNATIONAL JOURNAL OF MEDICINE, vol. 106, no. 6, 1 June 2013 (2013-06-01), pages 505 - 515, XP093116264, ISSN: 1460-2725, DOI: 10.1093/qjmed/hct051 *
ANONYMOUS: "Intrinsic Factor IgG ELISA Kit, Qualitative/semi-quantitative assay for Intrinsic Factor IgG antibodies", GENESIS DIAGNOSTICS, 18 August 2017 (2017-08-18), XP093116259, Retrieved from the Internet <URL:https://www.omegadx.com/Portals/0/GD035%20Intrinsic%20Factor%20IgG%20IFU%20-%20IVD.pdf> [retrieved on 20240105] *
BACON P A, GOLDBERG L S, BLUESTONE R: "Experimental humoral and cellular immunity to hog intrinsic factor", CANCER RESEARCH, WILEY-BLACKWELL PUBLISHING LTD., GB, vol. 25, no. 3, 1 September 1973 (1973-09-01), GB , pages 509 - 515, XP093116260, ISSN: 0019-2805 *
GREIBE EVA, NEXO EBBA: "Development of a Sensitive ELISA for Gastric Intrinsic Factor and Detection of Intrinsic Factor Immunoreactivity in Human Serum", NUTRIENTS, M D P I AG, CH, vol. 14, no. 19, CH , pages 4043, XP093116268, ISSN: 2072-6643, DOI: 10.3390/nu14194043 *
SAMLOFF I. MICHAEL, TURNER MICHAEL D.: "Rabbit Blocking and Binding Antibodies to Human Intrinsic Factor and Intrinsic Factor-B12 Complex", THE JOURNAL OF IMMUNOLOGY, WILLIAMS & WILKINS CO., US, vol. 101, no. 3, 1 September 1968 (1968-09-01), US , pages 578 - 586, XP093116262, ISSN: 0022-1767, DOI: 10.4049/jimmunol.101.3.578 *
SCHADE STANLEY G., ABELS JOHANNES, SCHILLING ROBERT F., FEICK PATRICIA, MUCKERHEIDE MARY: "Studies on Antibody to Intrinsic Factor *", JOURNAL OF CLINICAL INVESTIGATION, vol. 46, no. 4, 1 April 1967 (1967-04-01), pages 615 - 620, XP093116257, ISSN: 0021-9738, DOI: 10.1172/JCI105563 *

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