Anti-porcine TCN1 monoclonal antibody and methods for producing and using the same

Abstract

To provide new and improved methods of estimating hog TCN1 and / or removing hog TCN1 from HF preparations.SOLUTION: Anti-hog TCN1 monoclonal antibodies are disclosed, along with epitopes recognized by the anti-hog TCN1 monoclonal antibodies. Also there are disclosed kits containing the monoclonal antibodies and methods of producing the antibodies. Further there are disclosed methods of using the monoclonal antibodies, such as (but not limited to) in methods of estimating and / or removing TCN1 from hog intrinsic factor preparations.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS / INCORPORATION-BY-REFERENCE STATES This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 884,711, filed August 9, 2019. The entire contents of the above-referenced patents / patent applications are expressly incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not applicable. [Background technology]

[0003] Cobalamin is an essential nutrient, a natural water-soluble vitamin of the B group that must bind to intrinsic factor for intestinal absorption. Vitamin B12 (cyanocobalamin) is required for hematopoiesis, neuronal metabolism, DNA and RNA production, and carbohydrate, fat, and protein metabolism. Vitamin B12 improves iron function in the metabolic cycle and assists folate in choline synthesis. Vitamin B12 metabolism is interconnected with folate metabolism, and vitamin B12 deficiency causes pernicious anemia, megaloblastic anemia, and neurological disorders.

[0004] Transcobalamin I (TCN1), also known as haptocorrin, R-factor, and R-protein, is a glycoprotein produced by the salivary glands of the mouth. In the body, TCN1 primarily serves to protect cobalamin (vitamin B12) from acid degradation in the stomach by producing the TCN1 / vitamin B12 complex. As the complex moves into the more neutral duodenum, pancreatic proteases degrade TCN1, thereby releasing free vitamin B12, which then binds to intrinsic factor for absorption by ileal enterocytes.

[0005] Hog Intrinsic Factor (HIF) preparations are typically used in diagnostic vitamin B12 assays. However, porcine TCN1 (also known as piglet (Pig) TCN1, swine (Swine) TCN1, or hog (Hog) R protein) is the major HIF-related protein contaminant in crude extracts of porcine gastric mucosa, and the presence of porcine TCN1 in HIF preparations can compromise the integrity of any B12 assays in which HIF preparations are used. Therefore, it is essential that any TCN1 present in HIF preparations be estimated and / or removed. Summary of the Invention [Problem to be solved by the invention]

[0006] However, there are currently no commercial sources of anti-porcine TCN1 monoclonal antibodies available to estimate and / or remove porcine TCN1 from HIF preparations used in B12 assays. [Means for solving the problem]

[0007] Thus, there is a need in the art for anti-porcine TCN1 monoclonal antibodies and new and improved methods for estimating and / or removing porcine TCN1 from HIF preparations that overcome the shortcomings and drawbacks of the prior art. Directed to the present disclosure are such antibodies, kits containing the antibodies, and methods of making and using the antibodies. [Brief explanation of the drawings]

[0008] [Figure 1]Figure 1 shows the total fraction of vitamin B12-binding protein purified from a crude powder extract of porcine gastric mucosa on a column of vitamin B12-Sepharose according to Allen et al. (J. Biol. Chem. (1973) 248(10):3670-3680) and referred to for purposes of this disclosure as "partially purified porcine R protein." Panel A: SDS-PAGE under reducing conditions shows that the fraction contains native porcine R protein (broad 70-92 kDa band) and porcine intrinsic factor (narrow 55 kDa band). Panel B: Isoelectric focusing demonstrates that the isoelectric points of both glycosylated proteins—porcine R protein and porcine intrinsic factor—are nearly identical (pI<4.5). [Figure 2] Figure 1 shows an amino acid sequence alignment of transcobalamin I (TCN1) (SEQ ID NO: 1) and intrinsic factor (HIF) (SEQ ID NO: 17) from boar. Peptides 29-39 (also referred to herein as R1 and designated as SEQ ID NO: 2), peptides 80-89 (also referred to herein as R2 and designated as SEQ ID NO: 3), and peptides 200-215 (also referred to herein as R3 and designated as SEQ ID NO: 4), which do not share homology with porcine intrinsic factor, were selected for monoclonal antibody generation. [Figure 3] The locations of peptides R1, R2, and R3 on the surface of the protein globule are shown in front (Panel A) and top (Panel B) views of the model TCN1 crystal structure (PDB 4KKJ). [Figure 4] Immune sera from A / J mice immunized with different porcine TCN1 peptide-BSA conjugates were titrated on ELISA plates coated with synthetic peptides, peptide-OVA conjugates, or partially purified native porcine R proteins. As can be seen, all animals developed high antibody titers against the respective peptides and peptide-OVA conjugates; however, only sera from R1-BSA and R2-BSA animals bound partially purified native porcine R proteins. [Figure 5]Figure 1 shows the antigen-binding properties of 171B 1G5 mAb. This monoclonal antibody was generated against the R2-BSA conjugate. As can be seen, 171B 1G5 mAb is highly specific for the R2 peptide and recognizes both native and recombinant porcine TCN1 proteins. No cross-reactivity to porcine intrinsic factor was observed. [Figure 6] Immune sera from Balb / C (A) and A / J (B) mice immunized with partially purified native porcine R proteins were titrated on ELISA plates coated with synthetic peptides or partially purified native porcine R proteins. As can be seen, both mouse strains developed high antibody titers against porcine TCN1, but not against the synthetic R1, R2, or R3 peptides. [Figure 7] Figure 1 shows selection of hybridomas producing monoclonal antibodies against native pig TCN1. Hybridoma supernatants were screened by ELISA for binding of both rec piglet TCN1 and partially purified native pig R protein. As can be seen, monoclonal antibodies produced by the 171J 3F1 and 171J 3A6 clones bound rec piglet (Pig) TCN1 much better than native pig R protein; monoclonal antibody 171J 9G7 recognized both antigens equally well, and 171J 5H12 mAb bound native pig R protein more strongly than recombinant R protein. No cross-reactivity to porcine intrinsic factor was observed. [Figure 8] Figure 1 shows a summary of epitope mapping of mouse monoclonal antibodies assayed against a porcine TCN1 peptide microarray. Epitope mapping results confirmed the specificity of the mAbs to porcine TCN1 and identified linear epitopes unique to the 171B 1G5, 171J 3F1, 171J 3A6, and 171J 5H12 antibodies. Monoclonal antibody 171J 9G7 did not bind any linear porcine TCN1 or porcine intrinsic factor peptides. [Figure 9]The surface locations of the linear epitopes of the 171B 1G5, 171J 3F1, 171J 3A6, and 171J 5H12 antibodies are shown in front (Panel A) and top (Panel B) views of the model TCN1 crystal structure (PDB 4KKJ). [Figure 10] Figure 1 shows ELISA titration results of recombinant piglet TCN1 using conventionally produced 171B 1G5 monoclonal antibody versus recombinantly produced 171B 1G5 monoclonal antibody. As can be seen, the two titration curves are superimposable, and the data confirm the correct sequencing of the monoclonal antibody. [Figure 11] Native porcine R protein was affinity purified from a powdered crude extract of porcine gastric mucosa using a column with 171J 5H12 mAb. Panel A: SDS-PAGE under reducing conditions stained with Coomassie Blue R shows that the native porcine R protein sample (broad 70-92 kDa band) is homogeneous. Panel B: Isoelectric focusing with BioSafe Coomassie staining shows that native porcine TCN1 is a highly acidic protein with a pI of <4.5. [Figure 12] 1 shows the detection of native porcine TCN1 in crude extracts of porcine gastric mucosa using an ELISA assay with monoclonal antibody 171J 5H12. The detection limit is at least 1 ng / mL. [Figure 13] Porcine intrinsic factor sample polishing with 171J 5H12 mAb-Sepharose. HIF samples before and after depletion with affinity resin using 171J 5H12 monoclonal antibody were tested for the presence of native porcine TCN1 in an ELISA assay. As can be seen, after depletion, the level of porcine TCN1 contamination was significantly reduced from 2.7% to 0.1% of total protein. DETAILED DESCRIPTION OF THE INVENTION

[0009] Before describing at least one embodiment of the present disclosure in detail with illustrative terms and results, it should be understood that the disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description. The present disclosure is capable of other embodiments or of being practiced or carried out in various ways. Therefore, the phrases used herein are intended to have the broadest possible scope and meaning; and the embodiments are intended to be illustrative, not exhaustive. Also, it should be understood that the phraseology and terminology used herein are for the purpose of description and should not be regarded as limiting.

[0010] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature utilized in connection with, and the techniques of, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The techniques and procedures described above are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)) and Coligan et al., Current Protocols in Immunology (Current Protocols, Wiley Interscience (1994)), which are incorporated herein by reference. The nomenclature utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparations, formulations, and delivery, and treatment of patients.

[0011] All patents, published patent applications, and non-patent publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All patents, published patent applications, and non-patent publications referenced in any part of this application are expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0012] All of the compositions, kits, and / or methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions, kits, and / or methods have been described with reference to specific embodiments, it will be apparent to those skilled in the art that variations may be applied to the compositions, kits, and / or methods, and in the steps or sequence of steps of the methods described herein, without departing from the concept, spirit, and scope of the present disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present disclosure as defined by the appended claims.

[0013] As utilized in accordance with this disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0014] The use of the terms "a" or "an," when used in conjunction with the term "comprising" in the claims and / or this specification, can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one." Thus, the terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "compound" can refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or a greater number of compounds. The term "plurality" refers to two or more.

[0015] Use of the term "at least one" will be understood to include one and any amount greater than one, including, but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend to 100 or 1000 or more, depending on the term to which it is connected; furthermore, an amount of 100 / 1000 should not be considered limiting, as higher upper limits may also produce satisfactory results. Furthermore, use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. Use of ordinal terms (i.e., "first," "second," "third," "fourth," etc.) is only to distinguish between two or more items and is not meant to imply, for example, any sequence or order or importance of one item relative to another, or any order of addition.

[0016] Use of the term "or" in the claims is used to mean an inclusive "and / or" unless expressly indicated to refer to alternatives only or unless the alternatives are mutually exclusive. For example, the condition "A or B" is satisfied by any of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and A and B are both true (or exist).

[0017] As used herein, terms such as "one embodiment," "an embodiment," "some embodiments," "one example," "for example," and "the like" are used interchangeably. Any reference to "an example" means that a particular element, configuration, structure, or feature described in connection with an embodiment is included in at least one embodiment. The appearances of the phrases "in some embodiments" or "one example" in various places throughout this specification do not necessarily all refer to the same embodiment, for example. Moreover, any reference to one or more embodiments or examples should not be construed as a limitation on the scope of the claims.

[0018] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for a composition / apparatus / device, the method used to determine the value, or the variation that exists among test subjects. For example, but not by way of limitation, when the term "about" is used, the specified value may vary from the stated value by plus or minus 20 percent, or 15 percent, or 12 percent, or 11 percent, or 10 percent, or 9 percent, or 8 percent, or 7 percent, or 6 percent, or 5 percent, or 4 percent, or 3 percent, or 2 percent, or 1 percent, as such variations are appropriate for performing the disclosed method and would be understood by one of ordinary skill in the art.

[0019] As used in this specification and claims, the words "comprising" (and any form of including, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0020] The term "or combinations thereof," as used herein, refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and, if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations containing one or more repeats of an item or term are explicitly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those of skill in the art will understand that there is typically no limit to the number of items or terms in any combination unless otherwise apparent from the context.

[0021] As used herein, the term "substantially" means that the subsequently described event or circumstance occurs completely, or that the subsequently described event or circumstance occurs to a significant extent or degree. For example, when relating to a particular event or circumstance, the term "substantially" means that the subsequently described event or circumstance occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. The term "substantially adjacent" can mean that two items are 100% adjacent to each other, or that two items are within close proximity to each other but not 100% adjacent to each other, or that a portion of one of two items is within close proximity to the other item but not 100% adjacent to the other item.

[0022] The terms "analog" and "derivative" are used interchangeably herein to refer to a substance that contains the same basic carbon skeleton and carbon functionality in its structure as a given compound, but that may additionally contain one or more substitutions. The term "substituted" as used herein , will be understood to refer to the replacement of at least one substituent of a compound with a residue R. In certain non-limiting embodiments, R is selected from H, hydroxyl, thiol, halide (selected from fluoride, chloride, bromide, or iodide), C1-C4 compounds (selected from one of the following: optionally substituted linear, branched, or cyclic alkyl, and linear, branched, or cyclic alkenyl, where the optional substituents are selected from one or more of alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocycloalkyl). ), each of which is optionally substituted, the optional substituents being selected from one or more of alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, alkylaryl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocycloalkyl, phenyl, cyano, hydroxyl, alkyl, aryl, cycloalkyl, cyano, alkoxy, alkylthio, amino, —NH(alkyl), —NH(cycloalkyl), carboxy, and —C(O)-alkyl.

[0023] The term "sample," as used herein, will be understood to include any type of biological sample utilized in accordance with the present disclosure. Examples of fluid biological samples utilized include, but are not limited to, whole blood or any fraction thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, peritoneal fluid, cyst fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, feces, pleural effusion, nasopharyngeal fluid, combinations thereof, and the like.

[0024] The term "specific binding partner," as used herein, will be understood to refer to any molecule that can specifically bind to TCN1 for the purpose of detecting it. For example, but not by way of limitation, a specific binding partner may be an antibody, a receptor, a ligand, an aptamer, a molecularly imprinted polymer (i.e., an inorganic matrix), or any combination and / or derivative thereof, as well as any other molecule that can specifically bind to a macrophilin-binding pharmaceutical.

[0025] The terms "peptide," "polypeptide," and "protein" are used herein to refer to a polymer of amino acid residues. The term "polypeptide," as used herein, is a generic term referring to naturally occurring proteins, protein fragments, or analogs of a polypeptide sequence. Thus, naturally occurring proteins, protein fragments, and analogs are species of the polypeptide genus. The term "isolated peptide / polypeptide / protein," as used herein, refers to a peptide / polypeptide / protein of cDNA, recombinant RNA, or synthetic origin or some combination thereof; depending on its origin or derivation, an "isolated peptide / polypeptide / protein" is: (1) not associated with other peptides / polypeptides / proteins found in nature; (2) free of other peptides / polypeptides / proteins from the same source, e.g., free of murine proteins; (3) expressed by cells from a different species; and / or (4) not naturally occurring.

[0026] As used herein, the term "amino acid" includes all molecules, whether natural or synthetic, that contain both amino and acid functionalities and that can be included in a polymer of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; analogs, derivatives, and congeners thereof; amino acid analogs with variant side chains; and all stereoisomers of any of the foregoing.

[0027] The terms "polynucleotide" and "nucleic acid" are used interchangeably. They refer to any nucleic acid, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides refer to polymeric forms of nucleotides of a length of 1000 bp. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified, such as by conjugation with a labeling component. The terms "isolated nucleic acid" and "isolated polynucleotide" are used interchangeably; a nucleic acid or polynucleotide is considered "isolated" if it: (1) is not associated with all or a portion of a polynucleotide with which the "isolated polynucleotide" is found in nature, (2) is linked to a polynucleotide with which it is not naturally linked, or (3) does not occur in nature as part of a longer sequence.

[0028] The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments are ligated. Another type of vector is a viral vector, in which additional DNA segments are ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of the host cell upon introduction into the host cell, thereby replicating along with the host genome. Additionally, certain vectors are capable of driving the expression of genes. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").

[0029] The term "naturally occurring" as used herein when applied to an object refers to the fact that the object is found in nature.For example, the polynucleotide or polypeptide sequence that exists in organisms (including viruses) that can be isolated from natural sources and is not intentionally or otherwise modified by humans in the laboratory is naturally occurring.The term "naturally occurring" is used herein interchangeably with the term "native".

[0030] The term "selectively hybridize" as used herein means to detectably and specifically bind. Polynucleotides, oligonucleotides, and fragments thereof encoding peptides / polypeptides / proteins according to the present invention selectively hybridize to nucleic acid strands under hybridization and washing conditions that minimize detectable binding to nonspecific nucleic acids. High stringency conditions are used to achieve selective hybridization conditions known in the art and discussed herein. Generally, the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments according to the present invention and the nucleic acid sequence of interest will be at least 80%, more typically at least 85%, 90%, 95%, 99%, and 100%, with increasing homology. Two amino acid sequences are homologous if there is partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum correspondence. Gaps (in either of the two sequences being matched) are allowed to maximize correspondence; a gap length of 5 or less is preferred (but not limited), and 2 or less is more preferred (but not limited). Alternatively, two protein sequences (or polypeptide sequences derived therefrom of at least 30 amino acids in length) can be matched using the program ALIGN (standard) with a mutation data matrix and a gap penalty of 6 or more. Sequences are homologous (as that term is used herein) if they have an alignment score (in deviation units) greater than 5. See Dayhoff, MO, Atlas of Protein Sequence and Structure, pp. 101-110 (Vol. 5, National Biomedical Research Foundation (1972)) and Supplement 2 to this volume, pp. 1-10. Two sequences, or portions thereof, are more preferably homologous if their amino acids are 50% or more identical when optimally aligned using the ALIGN program. The term "corresponding to" is used herein to mean that a polynucleotide sequence is homologous to all or a portion of a reference polynucleotide sequence (i.e., identical, but not strictly evolutionarily related), or that a polypeptide sequence is identical to a reference polypeptide sequence. In contrast, the term "complementary to" is used herein to mean that a complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. By way of example, the nucleotide sequence "TATAC" corresponds to the reference sequence "TATAC" and is complementary to the reference sequence "GTATA."

[0031] The following terms are used to describe sequence relationships between two or more polynucleotide or amino acid sequences: "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." A "reference sequence" is a defined sequence used as a basis for sequence comparison; a reference sequence may be a subset of a larger sequence (e.g., as a segment of a full-length cDNA or gene sequence set forth in a sequence listing) or may comprise the entire cDNA or gene sequence. Generally, a reference sequence is at least 18 nucleotides or 6 amino acids in length, frequently at least 24 nucleotides or 8 amino acids in length, and often at least 48 nucleotides or 16 amino acids in length. Because two polynucleotide or amino acid sequences may each (1) contain similar sequences between the two molecules (i.e., portions of the complete polynucleotide or amino acid sequence) and (2) further contain sequences that differ between the two polynucleotide or amino acid sequences, sequence comparison between two (or more) molecules is typically performed by comparing the sequences of the two molecules over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window," as used herein, refers to a conceptual segment of at least 18 contiguous nucleotide positions or 6 amino acids, in which a polynucleotide sequence or amino acid sequence is compared to a reference sequence of at least 18 contiguous nucleotides or 6 amino acids, and the portion of the polynucleotide sequence in the comparison window may contain no more than 20 percent additions, deletions, substitutions, etc. (i.e., gaps) as compared to the reference sequence (no additions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences to align a comparison window can be performed by the local homology algorithm of Smith and Waterman (Adv. Appl. Math., 2:482 (1981)), the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol., 48:443 (1970)), the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. (USA), 85:2444 (1988)), computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, Geneworks, or MacVector software packages in the Wisconsin Genetics Software Package Release 7.0 (Genetics Computer Group, 575 Science Dr., Madison, Wis.)), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) produced by the various methods is selected.

[0032] The term "sequence identity" means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide-by-nucleotide or residue-by-residue basis) over the comparison window. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where an identical nucleic acid base (e.g., A, T, C, G, U, or I) or residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. The term "substantial identity," as used herein, refers to a characteristic of a polynucleotide or amino acid sequence, including sequences having at least 85 percent sequence identity, e.g., at least 90-95 percent sequence identity, or at least 99 percent sequence identity, relative to a reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions, frequently a window of at least 24-48 nucleotide (8-16 amino acid) positions, where the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that may contain deletions or additions totaling no more than 20 percent of the reference sequence over the comparison window. The reference sequence may be a subset of a larger sequence.

[0033] As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2nd ed., E.S. Golub and D.R. Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)), incorporated herein by reference. Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), α,α-disubstituted amino acids, unnatural amino acids such as N-alkylamino acids, lactic acid, and other unconventional amino acids may also be suitable components of the polypeptides of the present disclosure. Examples of unconventional amino acids include norleucine, 4-hydroxyproline, α-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0034] The term "substantial identity," as applied to polypeptides, means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, e.g., at least 90 percent sequence identity, or at least 95 percent sequence identity, or at least 99 percent sequence identity. In certain (but non-limiting) embodiments, residue positions that are not identical differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, the group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids having aliphatic hydroxyl side chains is serine and threonine; the group of amino acids having amide-containing side chains is asparagine and glutamine; the group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids having basic side chains is lysine, arginine, and histidine; and the group of amino acids having sulfur-containing side chains is cysteine ​​and methionine. Particular conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0035] The term "variant" of a reference polypeptide refers to a polypeptide that has one or more amino acid substitutions, deletions, or insertions compared to the reference polypeptide. Amino acid substitutions may be "conservative" or "non-conservative." A "conservative" amino acid substitution is one in which another amino acid has similar properties, such as, but not limited to, size and charge. Conservative substitutions refer to the substitution of amino acids in a polypeptide by a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally classified into families: (1) acidic = aspartic acid, glutamic acid; (2) basic = lysine, arginine, histidine; (3) nonpolar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More specifically, the families are: serine and threonine are the aliphatic hydroxy family; asparagine and glutamine are the amide-containing family; alanine, valine, leucine, and isoleucine are the aliphatic family; and phenylalanine, tryptophan, and tyrosine are the aromatic family. For example, it is reasonable to expect that isolated substitutions of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar substitutions of amino acids with structurally related amino acids will not significantly affect the binding or properties of the resulting molecule, particularly if the substitution does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. Fragments or analogs of antibody or immunoglobulin molecules are readily produced by those skilled in the art. Preferred amino and carboxy termini of fragments or analogs occur near the boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data to public or proprietary sequence databases. In certain (but non-limiting) embodiments, computer comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known (Bowie et al., Science, 253:164 (1991)).Thus, the above examples demonstrate that one of skill in the art can recognize sequence motifs and structural conformations that are used to define structural and functional domains according to the present disclosure.

[0036] Preferred amino acid substitutions are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and (5) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various mutations of sequences other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (such as, but not limited to, conservative amino acid substitutions) are made in the naturally occurring sequence (such as, but not limited to, portions of the polypeptide outside the domains that form intermolecular contacts). Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., the substituted amino acids should not tend to disrupt helices occurring in the parent sequence or other types of secondary structure that characterize the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, ed., W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure © (Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al. (Nature 354:105 (1991)), each of which is incorporated herein by reference.

[0037] The term "polypeptide fragment," as used herein, refers to a polypeptide that has an amino- and / or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally occurring sequence. A polypeptide fragment may be of any length, up to and including the length of the reference polypeptide.

[0038] The term "antibody" is used in the broadest sense and specifically (but not by way of limitation) encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), fragments of any of the above, and conjugates of any of the above, so long as they exhibit the desired biological activity of analyte binding. Thus, the term "antibody" or "antibody peptide" refers to a full-length immunoglobulin molecule (i.e., an intact antibody), or an antigen-binding fragment thereof that competes with the intact antibody for specific antigen binding. Antigen-binding fragments are produced by recombinant DNA methods or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include Fab, Fab', F(ab')2, Fv, scFv, disulfide-linked Fv, Fd, diabodies, single-chain antibodies, single-domain antibodies (such as, but not limited to, NANOBODIES®), and other antibody fragments and conjugates thereof that retain at least a portion of the variable region of an intact antibody, antibody surrogate proteins or peptides (i.e., engineered binding proteins / peptides), and combinations or derivatives thereof. See, for example, Hudson et al. (Nature Med. (2003) 9:129-134). Antibodies may be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0039] The term "antigen-binding fragment" or "antigen-binding portion" of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to bind to an antigen. The antigen-binding function of an antibody is performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, disulfide-linked Fv, Fd, diabodies, single-chain antibodies, single-domain antibodies (such as, but not limited to, NANOBODIES®), isolated CDRH3, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. These antibody fragments can be obtained using conventional recombinant and / or enzymatic methods and screened for antigen binding in the same manner as intact antibodies.

[0040] "Antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0041] "Antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. Kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0042] The term "CDR," and its plural "CDRs," refers to the complementarity-determining regions (CDRs) of an antibody or antibody fragment, which determine the binding characteristics of the antibody or antibody fragment. In most cases, three CDRs are present in the light chain variable region (CDRL1, CDRL2, and CDRL3) and three CDRs are present in the heavy chain variable region (CDRH1, CDRH2, and CDRH3). CDRs contribute to the functional activity of the antibody molecule and are separated by amino acid sequences comprising scaffolding or framework regions. Among the various CDRs, CDR3 sequences, particularly CDRH3, are the most diverse and therefore contribute most strongly to antibody specificity. There are at least two approaches to determining CDRs: (1) an approach based on cross-species sequence diversity (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. (1987)), which is incorporated by reference in its entirety); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Chothia et al., Nature, 342:877 (1989)), which is incorporated by reference in its entirety).

[0043] The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, an epitope is a region of an antigen that is specifically bound by an antibody. Epitopic determinants typically include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups. In certain embodiments, an epitope may have specific three-dimensional structural characteristics (e.g., a "conformational epitope") and specific charge characteristics.

[0044] An epitope is defined as "identical" to another epitope if a particular antibody specifically binds to both epitopes. In certain embodiments, polypeptides with different primary amino acid sequences may contain identical epitopes. In certain embodiments, identical epitopes may have different primary amino acid sequences. Different antibodies are said to bind to the same epitope when they compete for specific binding to the same epitope.

[0045] An antibody "specifically binds" an antigen if it preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules. In certain embodiments, an antibody comprises an antigen-binding site that specifically binds to a particular epitope. In certain such embodiments, an antibody can bind different antigens, so long as the different antigens contain that particular epitope or a closely related epitope. In certain cases, for example, homologous proteins from different species may contain the same epitope. In certain embodiments, an antibody may bind to a specific antigen, such as a 10 -6 M, 10 -7 M, 10 -8 M or 10 -9 Specifically binds to an antigen with a dissociation constant of less than or equal to M. When an antibody specifically binds to a receptor or ligand (i.e., a counter-receptor), the antibody can substantially inhibit adhesion of the receptor to the ligand. As used herein, an antibody substantially inhibits adhesion of a receptor to a ligand when excess antibody reduces the amount of receptor bound to the ligand by at least about 20%, 40%, 60%, or 80%, 85%, or 90% (as measured in an in vitro competitive binding assay).

[0046] An "isolated" antibody is one that has been separated and / or recovered from components of its production environment. Contaminating components of its production environment are substances that would interfere with diagnostic or therapeutic uses and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody will be measurably purified by at least three different methods: 1) to greater than 50% by weight of the antibody, e.g., greater than 75%, or greater than 85%, or greater than 95%, or greater than 99% by weight, as determined by the Lowry method; 2) to a degree sufficient to obtain at least 10 residues of N-terminal or internal amino acid sequence, e.g., at least 15 residues of sequence, using a spinning cup sequenator; or 3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or alternatively silver staining. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the environment in which the antibody is produced is absent. Ordinarily, however, isolated antibodies will be prepared by at least one purification step. Moreover, an "isolated antibody" is substantially free of other antibodies having different antigenic specificities, although an isolated antibody may have some cross-reactivity to other related antigens.

[0047] The term "antibody mutant" refers to an amino acid sequence variant of an antibody in which one or more amino acid residues have been modified. Such variants necessarily have less than 100% sequence identity or similarity with an amino acid sequence that has at least 75%, e.g., at least 80%, or at least 85%, or at least 90%, or at least 95% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of the antibody.

[0048] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a substantially homogeneous population of antibodies that specifically bind to the same epitope. That is, the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. In contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by a single production method, by a hybridoma culture, and thus are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, in one embodiment, monoclonal antibodies produced according to the present disclosure can be produced by the hybridoma method first described by Kohler and Milstein (Nature, 256:495 (1975)).

[0049] Monoclonal antibodies utilized in accordance with the present disclosure can be produced by any methodology known in the art, including, but not limited to, the result of a deliberate immunization protocol; the result of an immune response that naturally leads to the production of antibodies during the course of disease or cancer; phage-derived antibodies, etc. In addition to the hybridoma production methods listed above, monoclonal antibodies of the present disclosure can be produced by a variety of other methods, including, but not limited to, recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567); isolation of antibody fragments from phage display libraries (see, e.g., Clackson et al., Nature (1991) 352:624-628; and Marks et al., J. Mol. Biol. (1991) 222:581-597); and various other monoclonal antibody production techniques (see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)).

[0050] Once antibodies are obtained, e.g., individual B cells are identified and / or monoclonal antibodies are produced, sequences encoding the variable regions of these antibodies are obtained. Variable region sequences can be obtained, for example, by first sequencing antibody proteins produced by hybridomas, B cells, or phages to determine the encoding nucleic acid sequence. In one embodiment, immunoglobulin variable region (VH and VL) DNA or cDNA is instead sequenced. If the antibody is derived from a hybridoma cell line or isolated B cells, cDNA encoding the variable regions can be amplified using PCR, for example, by the methods described in Babcook et al. (Proc. Natl. Acad. Sci. USA, 93:7843-7848 (1996)) and PCT Publication No. WO 92 / 02551. The contents of both references are incorporated herein by reference in their entirety.

[0051] The term "neutralizing antibody" or "neutralizing antibody" refers to an antibody that reduces at least one activity of a polypeptide that contains an epitope to which the antibody specifically binds. In certain embodiments, a neutralizing antibody reduces an activity in vitro and / or in vivo.

[0052] The term "antigen-binding site" refers to a portion of an antibody capable of specifically binding an antigen. In certain embodiments, the antigen-binding site is provided by one or more antibody variable regions.

[0053] As used herein, "substantially pure" means that the subject species is the predominant species present (i.e., on a molar basis, the subject species is more abundant than any other individual species in the composition). Generally, a substantially pure composition will be one in which all of the macromolecular species present in the composition are present in a molar amount. It will comprise greater than about 50%, e.g., greater than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 99%. In one embodiment, the target species is purified to essential homogeneity (no contaminating species can be detected in the composition by conventional detection methods), and the composition consists essentially of a single macromolecular species.

[0054] The term "agent" refers to a chemical substance, a mixture of chemical substances, a biological polymer, or an extract made from biological materials. In certain embodiments, the "agent" may be a monoclonal antibody according to the present disclosure.

[0055] The term "antagonist" refers to an agent that decreases the activity of a protein / enzyme. The term "agonist" refers to an agent that increases the activity of a protein / enzyme.

[0056] Turning now to the inventive concept, unique and specific epitopes on porcine transcobalamin I (TCN1) have been identified, and murine monoclonal antibodies have been generated against these epitopes and / or crude preparations of porcine TCN1. These antibodies have several potential uses, including (for example, but not by way of limitation): estimation of TCN1 in crude porcine intestinal wall preparations (commonly used in the isolation of porcine IF); and removal of TCN1 from porcine intrinsic factor (HIF) preparations (for example, but not by way of limitation, by use of affinity chromatography), since HIF preparations are commonly used in B12 assays.

[0057] Certain non-limiting embodiments of the present disclosure relate to an antibody or antigen-binding fragment thereof that specifically binds to porcine transcobalamin-1 (TCN1). In certain non-limiting embodiments, the antibody or antigen-binding fragment thereof specifically binds to an epitope of porcine TCN1 selected from at least a portion of at least one of SEQ ID NOs: 2, 3, 4, and / or 20-23.

[0058] Certain non-limiting embodiments of the present disclosure relate to an antibody or antigen-binding fragment thereof that specifically binds to porcine transcobalamin-1 (TCN1), wherein the antibody comprises one or more of: (i) a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 8; (ii) a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 9; (iii) a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 10; (iv) a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 14; (v) a light chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 15; and (vi) a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 16. In certain non-limiting embodiments, the antibody or antigen-binding fragment thereof comprises two, three, four, or five of the above (i) to (vi). In another specific non-limiting embodiment, the antibody or antigen-binding fragment thereof comprises all of the above (i) to (vi).

[0059] Certain non-limiting embodiments of the present disclosure relate to an antibody or antigen-binding fragment thereof that specifically binds to porcine transcobalamin-1 (TCN1), wherein the antibody comprises one or more of: (i) a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 26; (ii) a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 27; (iii) a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 28; (iv) a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 31; (v) a light chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 32; and (vi) a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 33. In certain non-limiting embodiments, the antibody or antigen-binding fragment thereof comprises two, three, four, or five of the above (i) to (vi). In another specific non-limiting embodiment, the antibody or antigen-binding fragment thereof comprises all of the above (i) to (vi).

[0060] Certain non-limiting embodiments of the present disclosure relate to an antibody or antigen-binding fragment thereof that specifically binds to porcine transcobalamin-1 (TCN1), wherein the antibody: (i) has the structure of SEQ ID NO: 38 (ii) a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 39; (iii) a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 40; (iv) a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 43; (v) a light chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 44; and (vi) a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 45. In certain non-limiting embodiments, the antibody or antigen-binding fragment thereof comprises two, three, four, or five of the above (i) to (vi). In another specific non-limiting embodiment, the antibody or antigen-binding fragment thereof comprises all of the above (i) to (vi).

[0061] Certain non-limiting embodiments of the present disclosure relate to an antibody or antigen-binding fragment thereof that specifically binds to porcine transcobalamin-1 (TCN1), wherein the antibody comprises one or more of: (i) a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 50; (ii) a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 51; (iii) a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 52; (iv) a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 55; (v) a light chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 56; and (vi) a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 57. In certain non-limiting embodiments, the antibody or antigen-binding fragment thereof comprises two, three, four, or five of the above (i) to (vi). In another specific non-limiting embodiment, the antibody or antigen-binding fragment thereof comprises all of the above (i) to (vi).

[0062] Certain non-limiting embodiments of the present disclosure relate to an antibody or antigen-binding fragment thereof that specifically binds to porcine transcobalamin-1 (TCN1), wherein the antibody comprises one or more of: (i) a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 62; (ii) a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 63; (iii) a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 64; (iv) a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 67; (v) a light chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 68; and (vi) a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 69. In certain non-limiting embodiments, the antibody or antigen-binding fragment thereof comprises two, three, four, or five of the above (i) to (vi). In another specific non-limiting embodiment, the antibody or antigen-binding fragment thereof comprises all of the above (i) to (vi).

[0063] Certain non-limiting embodiments of the present disclosure relate to an antibody or antigen-binding fragment thereof selected from the group consisting of (A) to (E): (A) is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 8, a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 9, a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 10, a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 14, a light chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 15, and a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 16; and (B) is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 26, a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 27, a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 28, a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 31, and a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 32. (C) is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 38, a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 39, a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 40, a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 43, a light chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 44, and a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 45; (D) is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 50, a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 51, a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 52, a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 55, a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 56 and (E) is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 62, a heavy chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 63, a heavy chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 64, a light chain variable region CDR1 having the amino acid sequence of SEQ ID NO: 67, a light chain variable region CDR2 having the amino acid sequence of SEQ ID NO: 68, and a light chain variable region CDR3 having the amino acid sequence of SEQ ID NO: 69.

[0064] The antibody or antigen-binding fragment thereof may specifically bind to any epitope of porcine TCN1. For example, but not limited to, the antibody or antigen-binding fragment thereof may specifically bind to any portion of the amino acid sequence set forth in SEQ ID NO: 1 or a functional equivalent thereof (such as, but not limited to, an amino acid sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to at least a portion of SEQ ID NO: 1).

[0065] In certain (but non-limiting) embodiments, the epitope of porcine TCN1 to which the antibody / functional fragment specifically binds comprises at least a portion of SEQ ID NO: 2. In other certain (but non-limiting) embodiments, the epitope of porcine TCN1 to which the antibody / functional fragment specifically binds comprises at least a portion of SEQ ID NO: 3. In yet other certain (but non-limiting) embodiments, the epitope of porcine TCN1 to which the antibody / functional fragment specifically binds comprises at least a portion of SEQ ID NO: 4. In yet other certain (but non-limiting) embodiments, the epitope of porcine TCN1 to which the antibody / functional fragment specifically binds comprises at least a portion of SEQ ID NO: 20. In yet other certain (but non-limiting) embodiments, the epitope of porcine TCN1 to which the antibody / functional fragment specifically binds comprises at least a portion of SEQ ID NO: 21. In yet other certain (but non-limiting) embodiments, the epitope of porcine TCN1 to which the antibody / functional fragment specifically binds comprises at least a portion of SEQ ID NO: 22. In yet another specific (but non-limiting) embodiment, the epitope of porcine TCN1 to which the antibody / functional fragment specifically binds comprises at least a portion of SEQ ID NO:23.

[0066] In certain (but non-limiting) embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region comprising an amino acid sequence that is at least about 70% identical to SEQ ID NO: 7, 25, 37, 49, or 61, for example (but not limited to) at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO: 7, 25, 37, 49, or 61.

[0067] In certain (but non-limiting) embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region comprising an amino acid sequence that differs from SEQ ID NO: 7, 25, 37, 49, or 61 by fewer than about 25 amino acids, fewer than about 24 amino acids, fewer than about 23 amino acids, fewer than about 22 amino acids, fewer than about 21 amino acids, fewer than about 20 amino acids, fewer than about 19 amino acids, fewer than about 18 amino acids, fewer than about 17 amino acids, fewer than about 16 amino acids, fewer than about 15 amino acids, fewer than about 14 amino acids, fewer than about 13 amino acids, fewer than about 12 amino acids, fewer than about 11 amino acids, fewer than about 10 amino acids, fewer than about 9 amino acids, fewer than about 8 amino acids, fewer than about 7 amino acids, fewer than about 6 amino acids, fewer than about 5 amino acids, fewer than about 4 amino acids, fewer than about 3 amino acids, fewer than about 2 amino acids, or fewer than about 1 amino acid.

[0068] In certain (but non-limiting) embodiments, instead of and / or in addition to the above embodiments, the antibody or antigen-binding fragment thereof has a light chain variable region comprising an amino acid sequence that is at least about 70% identical to SEQ ID NO: 13, 30, 42, 54, or 66, for example (but not limited to) at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO: 13, 30, 42, 54, or 66.

[0069] In certain (but non-limiting) embodiments, the antibody or antigen-binding fragment thereof has a light chain variable region comprising an amino acid sequence that differs from SEQ ID NO: 13, 30, 42, 54, or 66 by fewer than about 21 amino acids, fewer than about 20 amino acids, fewer than about 19 amino acids, fewer than about 18 amino acids, fewer than about 17 amino acids, fewer than about 16 amino acids, fewer than about 15 amino acids, fewer than about 14 amino acids, fewer than about 13 amino acids, fewer than about 12 amino acids, fewer than about 11 amino acids, fewer than about 10 amino acids, fewer than about 9 amino acids, fewer than about 8 amino acids, fewer than about 7 amino acids, fewer than about 6 amino acids, fewer than about 5 amino acids, fewer than about 4 amino acids, fewer than about 3 amino acids, fewer than about 2 amino acids, or fewer than about 1 amino acid.

[0070] In certain (but non-limiting) embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region comprising an amino acid sequence that is at least about 90% identical to SEQ ID NO: 7, 25, 37, 49, or 61, and / or the antibody or antigen-binding fragment thereof has a light chain variable region comprising an amino acid sequence that is at least about 90% identical to SEQ ID NO: 13, 30, 42, 54, or 66. In certain (but non-limiting) embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region comprising an amino acid sequence that differs by fewer than about 13 amino acids from SEQ ID NO: 7, 25, 37, 49, or 61, and / or a light chain variable region comprising an amino acid sequence that differs by fewer than about 12 amino acids from SEQ ID NO: 13, 30, 42, 54, or 66.

[0071] In another specific (but non-limiting) embodiment, the antibody or antigen-binding fragment thereof has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, 25, 37, 49, or 61, and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 13, 30, 42, 54, or 66, respectively.

[0072] In another specific (but non-limiting) embodiment, the antibody or antigen-binding fragment thereof has a heavy chain comprising an amino acid sequence that is at least about 70% identical to SEQ ID NO:6, for example (but not limited to) at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO:6.

[0073] In certain (but non-limiting) embodiments, the antibody or antigen-binding fragment thereof is a fragment of SEQ ID NO: 6 and less than about 100 amino acids, less than about 90 amino acids, less than about 80 amino acids, less than about 75 amino acids, less than about 70 amino acids, less than about 65 amino acids, less than about 60 amino acids, less than about 55 amino acids, less than about 50 amino acids, less than about 45 amino acids, less than about 40 amino acids, less than about 35 amino acids, less than about 30 amino acids, less than about 25 ... Less than about 24 amino acids, less than about 23 amino acids, less than about 22 amino acids, less than about 21 amino acids, less than about 20 amino acids, less than about 19 amino acids, less than about 18 amino acids, less than about 17 amino acids, less than about 16 amino acids, less than about 15 amino acids, less than about 14 amino acids, less than about 13 amino acids, less than about 12 amino acids, less than about 11 amino acids, less than about 10 amino acids, less than about 9 amino acids, less than about 8 amino acids, less than about 7 amino acids, less than about 6 amino acids have heavy chains that comprise amino acid sequences that differ by less than about 5 amino acids, less than about 4 amino acids, less than about 3 amino acids, less than about 2 amino acids, or less than about 1 amino acid.

[0074] In certain (but non-limiting) embodiments, and alternatively and / or in addition to the above embodiments, the antibody or antigen-binding fragment thereof has a light chain comprising an amino acid sequence that is at least about 70% identical to SEQ ID NO: 12, for example (but not limited to) at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO: 12.

[0075] In certain (but non-limiting) embodiments, the antibody or antigen-binding fragment thereof has a light chain comprising an amino acid sequence that differs from SEQ ID NO: 12 by fewer than about 45 amino acids, fewer than about 40 amino acids, fewer than about 35 amino acids, fewer than about 30 amino acids, fewer than about 25 amino acids, fewer than about 24 amino acids, fewer than about 23 amino acids, fewer than about 22 amino acids, fewer than about 21 amino acids, fewer than about 20 amino acids, fewer than about 19 amino acids, fewer than about 18 amino acids, fewer than about 17 amino acids, fewer than about 16 amino acids, fewer than about 15 amino acids, fewer than about 14 amino acids, fewer than about 13 amino acids, fewer than about 12 amino acids, fewer than about 11 amino acids, fewer than about 10 amino acids, fewer than about 9 amino acids, fewer than about 8 amino acids, fewer than about 7 amino acids, fewer than about 6 amino acids, fewer than about 5 amino acids, fewer than about 4 amino acids, fewer than about 3 amino acids, fewer than about 2 amino acids, or fewer than about 1 amino acid.

[0076] In certain (but non-limiting) embodiments, the antibody or antigen-binding fragment thereof has a heavy chain comprising an amino acid sequence that is at least about 90% identical to SEQ ID NO: 6, and / or the antibody or antigen-binding fragment thereof has a light chain comprising an amino acid sequence that is at least about 70% identical to SEQ ID NO: 12. In certain (but non-limiting) embodiments, the antibody or antigen-binding fragment thereof has a heavy chain comprising an amino acid sequence that differs by fewer than about 47 amino acids from SEQ ID NO: 6, and / or a light chain comprising an amino acid sequence that differs by fewer than about 24 amino acids from SEQ ID NO: 12.

[0077] In another specific (but non-limiting) embodiment, the antibody or antigen-binding fragment thereof has a heavy chain comprising the amino acid sequence of SEQ ID NO:6 and / or a light chain comprising the amino acid sequence of SEQ ID NO:12.

[0078] In yet another specific (but non-limiting) embodiment, the antibody or functional fragment has a heavy chain encoded by a polynucleotide that is at least about 70% identical to SEQ ID NO:5, or a heavy chain variable region encoded by a polynucleotide that is at least about 70% identical to SEQ ID NO:24, 36, 48, or 60, for example (but not limited to) at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO:5, 24, 36, 48, or 60.

[0079] In yet another specific (but non-limiting) embodiment, and alternatively and / or additionally to the above embodiments, the antibody or functional fragment has a light chain encoded by a polynucleotide that is at least about 70% identical to SEQ ID NO: 11, or at least about 70% identical to SEQ ID NO: 29, 41, 53, or 65, such as (but not limited to) at least about 75% identical to SEQ ID NO: 11, 29, 41, 53, or 65, and each of the above light chain variable regions has a light chain variable region encoded by a polynucleotide that is about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the above light chain variable region.

[0080] In further specific (but non-limiting) embodiments, the antibody or functional fragment has a heavy chain encoded by a polynucleotide at least about 70% identical to SEQ ID NO: 5, and / or the antibody or functional fragment has a light chain encoded by a polynucleotide at least about 70% identical to SEQ ID NO: 11. In further specific (but non-limiting) embodiments, the antibody or functional fragment has a heavy chain variable region encoded by a polynucleotide at least about 70% identical to SEQ ID NO: 24, 36, 48, or 60, and / or the antibody or functional fragment has a light chain variable region encoded by a polynucleotide at least about 70% identical to SEQ ID NO: 29, 41, 53, or 65, respectively.

[0081] In yet another specific (but non-limiting) embodiment, the antibody or antigen-binding fragment thereof has a heavy chain encoded by a sequence that differs from SEQ ID NO: 5, or a heavy chain variable region that differs from SEQ ID NO: 24, 36, 48, or 60 by fewer than about 100 nucleotides, fewer than about 90 nucleotides, fewer than about 80 nucleotides, fewer than about 75 nucleotides, fewer than about 70 nucleotides, fewer than about 60 nucleotides, fewer than about 50 nucleotides, fewer than about 45 nucleotides, fewer than about 40 nucleotides, fewer than about 35 nucleotides, fewer than about 30 nucleotides, fewer than about 225 nucleotides, fewer than about 20 nucleotides, fewer than about 15 nucleotides, fewer than about 10 nucleotides, fewer than about 9 nucleotides, fewer than about 8 nucleotides, fewer than about 7 nucleotides, fewer than about 6 nucleotides, fewer than about 5 nucleotides, fewer than about 4 nucleotides, fewer than about 3 nucleotides, fewer than about 2 nucleotides, or fewer than about 1 nucleotide.

[0082] In yet another specific (but non-limiting) embodiment, and instead and / or in addition to the above embodiments, the antibody or antigen-binding fragment thereof has a light chain encoded by a sequence that differs from SEQ ID NO: 11, or a light chain variable region that differs from SEQ ID NO: 30, 42, 54, or 66 by fewer than about 100 nucleotides, fewer than about 90 nucleotides, fewer than about 80 nucleotides, fewer than about 75 nucleotides, fewer than about 70 nucleotides, fewer than about 60 nucleotides, fewer than about 50 nucleotides, fewer than about 45 nucleotides, fewer than about 40 nucleotides, fewer than about 35 nucleotides, fewer than about 30 nucleotides, fewer than about 25 nucleotides, fewer than about 20 nucleotides, fewer than about 15 nucleotides, fewer than about 10 nucleotides, fewer than about 9 nucleotides, fewer than about 8 nucleotides, fewer than about 7 nucleotides, fewer than about 6 nucleotides, fewer than about 5 nucleotides, fewer than about 4 nucleotides, fewer than about 3 nucleotides, fewer than about 2 nucleotides, or fewer than about 1 nucleotide.

[0083] In yet another specific (but non-limiting) embodiment, the antibody or antigen-binding fragment thereof has a heavy chain encoded by a sequence that differs by fewer than about 100 nucleotides from SEQ ID NO: 5 and / or a light chain encoded by a sequence that differs by fewer than about 70 nucleotides from SEQ ID NO: 11. In yet another specific (but non-limiting) embodiment, the antibody or antigen-binding fragment thereof has a heavy chain variable region encoded by a sequence that differs by fewer than about 100 nucleotides from SEQ ID NO: 25, 37, 49, or 61, and / or a light chain variable region encoded by a sequence that differs by fewer than about 70 nucleotides from SEQ ID NO: 29, 41, 53, or 65, respectively.

[0084] Standard assays for assessing the binding ability of antibodies are known in the art, including, for example (but not limited to), ELISA, Western blot, and RIA, as well as other types of suitable assays known in the art. The binding kinetics (e.g., binding affinity) of antibodies can also be assessed by standard assays known in the art, such as (but not limited to) by Biacore analysis. In some non-limiting embodiments, the antibodies described herein are capable of binding at about 10 -6 M, about 10 -7 M, about 10 -8 M, about 10 -9 M, or about 10 -10 In one particular (but non-limiting) embodiment, the antibody binds to the above sequences with a dissociation constant of about 10 -7 It binds to an epitope on porcine TCN1 with a dissociation constant of less than M.

[0085] The antibody or antigen-binding fragment thereof may be a monoclonal antibody or antigen-binding fragment thereof. Alternatively, the antibody or antigen-binding fragment thereof may be a polyclonal antibody or antigen-binding fragment thereof.

[0086] In certain non-limiting embodiments, the antibody or antigen-binding fragment thereof is further defined as being selected from a full-length immunoglobulin molecule, an scFv, a Fab fragment, a Fab' fragment, F(ab')2, an Fv, a disulfide-linked Fv, and combinations thereof.

[0087] In certain non-limiting embodiments, the antibody or antigen-binding fragment thereof is isolated. In certain (but non-limiting) embodiments, the antibody or antigen-binding fragment thereof is purified.

[0088] The present disclosure also relates to antibodies or functional fragments thereof that bind to the same epitope as any of the antibodies or functional fragments described herein above.

[0089] Certain non-limiting embodiments of the present disclosure also relate to a method for producing an antibody or antigen-binding fragment thereof capable of specifically binding to an epitope of porcine transcobalamin-1 (TCN1), comprising the steps of immunizing a non-human animal with an antigenic compound comprising at least one peptide of SEQ ID NOs: 2-4 and / or 20-23; and recovering the antibody or antigen-binding fragment thereof from the plasma of the non-human animal.

[0090] Certain non-limiting embodiments of the present disclosure relate to hybridomas that produce any of the antibodies or antigen-binding fragments thereof described herein above.

[0091] Certain non-limiting embodiments of the present disclosure relate to methods for producing an antibody or antigen-binding fragment thereof capable of specifically binding to an epitope of porcine transcobalamin-1 (TCN1), in which a hybridoma described herein above is cultured to produce any of the antibodies or antigen-binding fragments described herein above. In at least certain non-limiting embodiments, the antibody or antigen-binding fragment thereof is recovered.

[0092] Certain non-limiting embodiments of the present disclosure also relate to conjugates comprising any of the antibodies or antigen-binding fragments thereof disclosed or otherwise contemplated herein attached to a detectable label. Non-limiting examples of detectable labels utilized by the present disclosure include enzyme labels, radioactive labels, fluorescent labels, chemiluminescent labels, bioluminescent labels, and particulate labels, as well as any combination thereof. Furthermore, the detectable label can be attached to the antibody or functional fragment via direct or indirect conjugation.

[0093] Certain non-limiting embodiments of the present disclosure also relate to conjugates comprising any of the antibodies or antigen-binding fragments thereof disclosed or otherwise contemplated herein attached to a solid support. Attachment of the antibody / antigen-binding fragment to the solid support (via direct or indirect conjugation) can be achieved, for example (but not limited to), by affinity chromatography, such as on a column. A purified chromatographic substrate is obtained.

[0094] Certain non-limiting embodiments of the present disclosure relate to polynucleotides encoding any of the antibodies or antigen-binding fragments thereof disclosed or otherwise contemplated herein.

[0095] In certain (but non-limiting) embodiments, a portion of the polynucleotide encoding the heavy chain or heavy chain variable region of the antibody or antigen-binding fragment thereof is at least about 70% identical to SEQ ID NO: 5, 24, 36, 48, or 60, for example (but not limited to) at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO: 5, 24, 36, 48, or 60.

[0096] In yet another specific (but non-limiting) embodiment, and instead and / or in addition to the above embodiments, the portion of the polynucleotide encoding the heavy chain or heavy chain variable region of the antibody or antigen-binding fragment thereof is at least about 70% identical to SEQ ID NO: 11, 29, 41, 53, or 65, for example (but not limited to) at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to SEQ ID NO: 11, 29, 41, 53, or 65.

[0097] In yet another specific (but non-limiting) embodiment, a portion of the polynucleotide encoding the heavy chain or heavy chain variable region of the antibody or antigen-binding fragment thereof is at least about 90% identical to SEQ ID NO: 5, 24, 36, 48, or 60, and / or a portion of the polynucleotide encoding the light chain or light chain variable region of the antibody or antigen-binding fragment thereof is at least about 90% identical to SEQ ID NO: 11, 29, 41, 53, or 65, respectively.

[0098] In further specific (but non-limiting) embodiments, a portion of the polynucleotide encoding the heavy chain or heavy chain variable region of the antibody or antigen-binding fragment thereof is at least about 90% identical to SEQ ID NO: 5, 24, 36, 48, or 60, and / or a portion of the polynucleotide encoding the light chain or light chain variable region of the antibody or antigen-binding fragment thereof is at least about 90% identical to SEQ ID NO: 11, 29, 41, 53, or 65, respectively.

[0099] In another specific (but non-limiting) embodiment, the portion of the sequence corresponding to the heavy chain or heavy chain variable region of the antibody or antigen-binding fragment thereof, and / or the portion of the sequence corresponding to the light chain or light chain variable region of the antibody or antigen-binding fragment thereof, is selected from the group consisting of SEQ ID NO: 5, 24, 36, 48, or 60 or SEQ ID NO: 11, 29, 41, 53, or 65, respectively, and less than about 100 nucleotides, less than about 90 nucleotides, less than about 80 nucleotides, less than about 75 nucleotides, less than about 70 nucleotides, less than about 60 nucleotides. The amino acid sequence differs by less than about 50 nucleotides, less than about 45 nucleotides, less than about 40 nucleotides, less than about 35 nucleotides, less than about 30 nucleotides, less than about 25 nucleotides, less than about 20 nucleotides, less than about 15 nucleotides, less than about 10 nucleotides, less than about 9 nucleotides, less than about 8 nucleotides, less than about 7 nucleotides, less than about 6 nucleotides, less than about 5 nucleotides, less than about 4 nucleotides, less than about 3 nucleotides, less than about 2 nucleotides, or less than about 1 nucleotide.

[0100] In yet another specific (but non-limiting) embodiment, the portion of the sequence corresponding to the heavy chain or heavy chain variable region of the antibody or antigen-binding fragment thereof differs by fewer than about 100 nucleotides from SEQ ID NO: 5, 24, 36, 48, or 60, and / or the portion of the sequence corresponding to the light chain or light chain variable region of the antibody or antigen-binding fragment thereof differs by fewer than about 70 nucleotides from SEQ ID NO: 11, 29, 41, 53, or 65.

[0101] Certain non-limiting embodiments of the present disclosure relate to vectors comprising any of the polynucleotides encoding the antibodies or antigen-binding fragments thereof described or otherwise contemplated herein.

[0102] Certain non-limiting embodiments of the present disclosure relate to recombinant host cells comprising any of the polynucleotides encoding the antibodies or antigen-binding fragments thereof described or otherwise contemplated herein. Certain non-limiting embodiments of the present disclosure relate to recombinant host cells comprising any of the vectors described or otherwise contemplated herein.

[0103] Certain non-limiting embodiments of the present disclosure relate to methods for making an antibody or antigen-binding fragment thereof capable of specifically binding to porcine transcobalamin-1 (TCN1), comprising: (a) culturing any of the recombinant host cells described or otherwise contemplated herein in cell culture under conditions that allow for expression of the antibody or antigen-binding fragment thereof encoded by the polynucleotide; and (b) isolating the antibody or antigen-binding fragment thereof from the cell culture.

[0104] Certain non-limiting embodiments of the present disclosure relate to methods for detecting TCN1 present in a porcine intrinsic factor (HIF) preparation, comprising: contacting the HIF preparation with any of the antibodies or antigen-binding fragments thereof disclosed or otherwise contemplated herein under conditions in which an antibody / TCN1 complex is formed if TCN1 is present in the HIF preparation; and detecting any antibody / TCN1 complex formed, wherein the amount of antibody-TCN1 complex formed is directly proportional to the amount of TCN1 present in the HIF preparation.

[0105] In a particular (but non-limiting) embodiment, the labels described herein above are attached to antibodies / functional fragments for use in detecting antibody / TCN1 complexes.

[0106] Certain non-limiting embodiments of the present disclosure relate to methods for removing TCN1 present in a porcine intrinsic factor (HIF) preparation, the method comprising: contacting the HIF preparation with any of the antibodies or antigen-binding fragments thereof disclosed or otherwise contemplated herein under conditions such that an antibody / TCN1 complex is formed if TCN1 is present in the HIF preparation; and removing the antibody / TCN1 complex from the HIF preparation.

[0107] In a specific (but non-limiting) embodiment, the antibody or antigen-binding fragment thereof is bound to a solid support, as described herein above, which aids in the removal of the antibody-TCN1 complex from the HIF preparation.

[0108] In certain (but non-limiting) embodiments, the method may further comprise eluting TCN1 from the antibody / TCN1 complex, thereby purifying TCN1 from the HIF preparation. Thus, the method for removing TCN1 present in a porcine intrinsic factor preparation also serves as a method for purifying TCN1 from a porcine intrinsic factor preparation.

[0109] Certain non-limiting embodiments of the present disclosure also include antibodies / antigen-binding fragments and / or compositions disclosed or otherwise contemplated herein (including those with detectable labels or solid supports). The present invention also includes kits containing any of the compositions (e.g., compositions comprising an antibody / antigen-binding fragment thereof) attached thereto, as well as any other reagents utilized in the assays / methods described herein and / or vitamin B12 assays performed after TCN1 has been deduced and / or removed from the HIF preparation. For example (and not by way of limitation), the kit may further comprise an antibody or antigen-binding fragment thereof that binds to HIF.

[0110] The assay components / reagents of the compositions / kits / methods are provided in any form that enables them to function according to the present disclosure. For example, but not by way of limitation, each of the reagents is provided in liquid form and disposed in the kit in bulk and / or single aliquot form. Alternatively, in certain (but non-limiting) embodiments, one or more of the reagents are disposed in the kit in the form of a single aliquot lyophilized reagent. The use of dried reagents in microfluidic devices is described in detail in U.S. Pat. No. 9,244,085 (Samproni), the entire contents of which are expressly incorporated herein by reference.

[0111] In addition to the assay components / reagents detailed herein above, the kit may further contain other reagents for carrying out any of the specific assays described or otherwise contemplated herein. The nature of these additional reagents will depend on the specific assay format, and their identification is well within the skill of those skilled in the art; therefore, further description thereof is deemed unnecessary. Additionally, the components / reagents present in the kit may each be contained in separate containers / compartments, or, depending on the cross-reactivity and stability of the components / reagents, various components / reagents may be combined in one or more containers / compartments. Additionally, the kit may include a microfluidic device in which the components / reagents are disposed.

[0112] The relative amounts of the various components / reagents in the kit can vary widely to provide concentrations of the components / reagents that substantially optimize the reactions required to occur during the assay method and further to substantially optimize the sensitivity of the assay. Under appropriate conditions, one or more of the components / reagents in the kit can be provided as a dry powder, such as a lyophilized powder, and the kit may further include an excipient for dissolving the dried reagent; in this manner, a reagent solution having the appropriate concentrations for performing the method or assay according to the present disclosure can be obtained from these components. Positive and / or negative controls can also be included with the kit. Furthermore, the kit may further include a set of written instructions describing how to use the kit. Kits of this nature can be used in any of the methods described or otherwise contemplated herein. [Example]

[0113] Examples are provided below. However, it should be understood that the present disclosure should not be limited in its application to the specific experiments, results, and experimental procedures disclosed herein. Rather, the examples are simply provided as one of various embodiments and are intended to be illustrative and not exhaustive. [Example]

[0114] Identification of epitopes of porcine TCN1 and generation of anti-porcine TCN1 monoclonal antibodies using synthetic peptides The porcine R protein (TCN1) is a glycoprotein containing 392 amino acids (including a polypeptide chain Mw of approximately 46 kDa) and up to approximately 50% w / w carbohydrate; its amino acid sequence is shown in SEQ ID NO: 1. TCN1 protects acid-sensitive vitamin B12 during its transit through the stomach.

[0115] For purposes of this disclosure, recombinant yeast-produced non-glycosylated piglet TCN1 protein with a Mw of 46.3 kDa (LifeSpan Biosciences, Inc., L S-G23154, Seattle, WA) is referred to herein as "rec piglet (Pig) TCN1," whereas glycosylated piglet TCN1 purified from a crude powder extract of pig gastric mucosa and having a Mw of approximately 70-92 kDa is referred to herein as "native piglet (Hog) R protein."

[0116] TCN1 and intrinsic factor (IF), as well as TCN2, all express K D They have exceptional affinity for the biologically active form of cobalamin (B12) at <1 pm; however, they each exhibit different selectivity for nonfunctional cobalamin analogs. IFs and, to some extent, TCNs, are sensitive to changes in ligand structure, which helps these proteins distinguish between physiologically active and inactive corrinoids. In contrast, TCN1 can successfully bind many truncated corrinoids that entirely lack the nucleotide moiety. Binding to the carrier shields the lower part of the cobalamin molecule, which contains the nucleotide (also called the α-site). In contrast, the upper surface of cobalamin (β-site), which bears the active group, appears open in the case of holoIFs and holoHCs, as judged by their reactivity with external compounds.

[0117] Porcine TCN1 is a major contaminant in porcine intrinsic factor (HIF) preparations used in vitamin B12 assays. Vitamin B12-binding proteins can be separated from most gastric proteins that do not bind vitamin B12 using affinity chromatography on vitamin B12-Sepharose, as described by Allen et al. (J. Biol. Chem. (1973) 248(10):3670-3680). Figure 1 shows the total fraction of vitamin B12-binding proteins purified from a crude powder extract of porcine gastric mucosa on a vitamin B12-Sepharose column. SDS-PAGE under reducing conditions (Panel A) shows that the fraction contains native porcine R protein (broad band between 70 and 92 kDa) and porcine intrinsic factor (narrow band at 55 kDa). Isoelectric focusing (Panel B) demonstrates that the isoelectric points of both proteins are nearly identical (pI < 4.5).

[0118] For the purposes of this disclosure, the total fraction of vitamin B12 binding protein purified from a crude powder extract of porcine gastric mucosa on a column of vitamin B12-Sepharose is referred to herein as "partially purified native porcine R protein."

[0119] As can be seen in Figure 2, which contains an amino acid sequence alignment of transcobalamin I (TCN1) and intrinsic factor (HIF) from boar, the polypeptide chains of the two proteins share 31% identity and 49% homology.

[0120] The two vitamin B12-binding proteins are extremely difficult to separate from each other by routine methods such as ammonium sulfate fractionation, size exclusion, or ion-exchange chromatography because the two proteins have similar molecular weights and nearly identical isoelectric points (Figure 1). Affinity chromatography on a column using a porcine TCN1-specific monoclonal antibody (mAb) would allow for the separation of porcine TCN1 and HIF.

[0121] To our knowledge, only one mouse monoclonal antibody, clone 3F10, is commercially available for human TCN1, and no monoclonal antibodies for porcine TCN1 or porcine intrinsic factor (HIF) are available. However, this anti-human TCN1 antibody does not cross-react with porcine TCN1.

[0122] Monoclonal antibodies specific for the native porcine R protein will allow the development of an ELISA to estimate porcine TCN1 in crude material and HIF preparations. Antibodies against porcine TCN1 would also allow for the removal of porcine TCN1 contaminants from HIF preparations and / or the development of affinity columns (or other affinity matrices) for use in polishing purified HIF, if necessary.

[0123] An affinity column using anti-porcine R protein monoclonal antibodies would enable the purification of native glycosylated porcine R proteins from natural sources. To our knowledge, only nonglycosylated yeast-produced recombinant piglet TCN1 protein is commercially available (LifeSpan Biosciences, Inc., LS-G23154, Seattle, WA); native porcine TCN1 is not currently available.

[0124] To identify unique and specific epitopes on porcine TCN1, an amino acid sequence alignment of TCN1 (or haptocorrin, accession number P17830.2) from wild boar and intrinsic factor (HIF, accession number XP_003122730.2) was performed using the Basic Local Alignment Search Tool (BLSAS). This was performed using BLASTA (Blockchain for Atomic Energy) software (National Center for Biotechnology Information, U.S. National Library of Medicine, Bethesda, MD). As shown in Figure 2, several porcine TCN1 peptides that share no homology with HIF were identified. These peptides were designated R1, R2, and R3. Their amino acid sequences are shown in Table 1.

[0125] The surface localization of the R1, R2, and R3 epitopes on the TCN1 molecule was confirmed using DeepView / Swiss-PdbViewer software from the Swiss Institute of Bioinformatics (Lausanne, Switzerland). The crystal structure of human haptocorrin in complex with cyanocobalamin at 2.35 Å resolution (PDB 4KKJ, Furger et al., J. Biol. Chem. (2013) 288(35):25466–25476) was used as a 3D model to identify the peptide locations. The locations of the R1, R2, and R3 peptides on the surface of the TCN1 molecule are shown in Figure 3, in front (Panel A) and top (Panel B) views of the model crystal structure.

[0126] Analysis of the amino acid sequences of the R1, R2, and R3 peptides by FASTA software (EMBL-EBI, Hinxton, Cambridge) confirmed that these peptides correspond to epitopes unique to the porcine R protein. Therefore, a specific mouse monoclonal antibody against one of the epitopes was subsequently developed.

[0127] [Table 1]

[0128] To generate monoclonal antibodies against the above-described epitopes, synthetic peptides and their respective conjugates with ovalbumin (OVA) and bovine serum albumin (BSA) were produced at Bio-Synthesis, Inc. (Lewisville, TX) as outlined in Table 2. Note that for peptide R2, two methionine residues present in the native porcine TCN1 sequence were modified to norleucine for synthesis of the peptide and its conjugates used as immunogens.

[0129] Antibodies were produced in accordance with federal regulations and IACUC protocols. The murine system has worked very well for developing antibodies for use in in vitro diagnostic applications and was therefore used to generate anti-porcine TCN1 antibodies.

[0130] [Table 2]

[0131] In this example, monoclonal antibodies against porcine TCN1 peptides were generated by intraperitoneally (IP) immunization of female BALB / c, Swiss Webster (SW), or A / J mice with immunogens containing the R1, R2, or R3 peptides conjugated to BSA. Mice received three or more injections of immunogen (50 μg / dose / animal) at 3-week intervals. Primary immunizations were performed using antigen emulsified in complete Freund's adjuvant (CFA), followed by subsequent booster immunizations in incomplete Freund's adjuvant (IFA).

[0132] One week after the final injection, mice were bled, and serum samples were tested for peptide-specific antibodies in an ELISA assay using the corresponding ovalbumin conjugate and free peptide. All ELISA steps were performed at room temperature. Nunc Maxi-Sorp™ flat-bottom ELISA plates were coated with 2 and 1 μg / mL of peptide or peptide-OVA conjugate in PBS, respectively, at 50 μL per well for 1 hour. The plates were tapped dry, and 200 μL of blocking solution (0.5% casein in PBS containing 0.05% Tween 20) was added per well to block remaining binding sites for 1 hour. The plates were washed three times with Milli-Q water containing 0.05% Tween 20, and the antibody samples to be tested (serum, hybridoma supernatant, or PBS antibody dilution) were added to the plate wells at a volume of 50 μL per well. After 1 hour of incubation, the plates were washed again as above, and goat anti-mouse IgG-HRP conjugate diluted 1:3,000 in blocking solution was added at 50 μL per well for 1 hour. The plates were washed, and TMB substrate (Moss, Pasadena, MD) was added at 100 μL per well for 15 minutes. The optical density of the samples was measured at 650 nm using an ELISA plate reader.

[0133] Serum samples were partially purified with vitamin B12-Sepharose and purified to remove contaminants from porcine intrinsic factor. The binding of purified native porcine R protein samples was also tested (Figure 1). For this purpose, ELISA plates were coated with 1 μg / mL partially purified native porcine R protein in PBS at 50 μL per well for 1 h, and subsequent ELISA steps were performed as described above.

[0134] Figure 4 shows the results of mouse blood titrations of selected A / J mice. Mice C3, C1, and C5 were immunized with R1-BSA, R2-BSA, and R3-BSA, respectively. As can be seen, all animals developed high antibody titers against the respective peptides and peptide-OVA conjugates, but only sera from R1-BSA and R2-BSA-immunized animals bound native porcine R proteins. These results confirm that the synthetic R1 and R2 peptides elicit an immune response against native porcine TCN1. Furthermore, the epitope corresponding to the synthetic R3 peptide may not be readily available for antibody binding on the native, heavily glycosylated protein.

[0135] Mice with high anti-peptide antibody titers were selected for monoclonal antibody generation. Mice received pre-fusion boosts with the same immunogen (25 μg / dose / animal in PBS, IP) for three consecutive days prior to fusion. On day 4, mice were sacrificed, and splenocytes were harvested. Immunized splenocytes and P3-X63Ag8.653 mouse myeloma cells (ATCC CRL-1580™) were fused in the presence of polyethylene glycol. The fused cells were suspended in HAT-containing medium and cultured for approximately 10–21 days. Hybridoma supernatants were screened for anti-peptide antibodies by ELISA using plates coated with each peptide as described above. Positive clones were subcloned by limiting dilution, expanded, and frozen. Monoclonal antibodies were purified from the hybridoma supernatants on a column of Protein A Sepharose (GE Healthcare, Chicago, IL). Table 3 shows the characteristics of several monoclonal antibodies generated against the porcine TCN1 peptide.

[0136] [Table 3]

[0137] The purified mAbs were tested in ELISA assays for binding to the corresponding peptides, partially purified native porcine R protein, and recombinant piglet TCN1 (LifeSpan BioSciences, LS-G23154, Seattle, WA), as well as porcine intrinsic factor. To this end, two-fold dilutions of antigen starting at 8 μg / mL in PBS were added to the wells of an ELISA plate at room temperature for 1 h at 50 μL per well. The plates were tapped dry, blocked with 200 μL of blocking solution per well, and washed three times as described above. A monoclonal antibody against the porcine TCN1 peptide at 1 μg / mL in PBS was added at 50 μL per well. After 1 h of incubation, the plates were washed again, and a goat anti-mouse IgG-HRP conjugate diluted 1:3,000 in blocking solution was added at 50 μL per well for 1 h. Plates were washed and TMB substrate (Moss, Pasadena, MD) was added at 100 μL per well for 15 minutes. Plates were read at 650 nm using an ELISA plate reader. .

[0138] All anti-peptide monoclonal antibodies demonstrated strong dose-dependent binding to the corresponding synthetic peptides, but only the 171B 1G5 mAb generated against the R2 peptide recognized both recombinant piglet TCN1 and partially purified native porcine R protein. Figure 5 shows the ELISA results of different antigen binding by the 171B 1G5 monoclonal antibody. As can be seen, the 171B 1G5 monoclonal antibody bound both the R2 peptide and native and recombinant porcine TCN1 proteins. The mAb also demonstrated no cross-reactivity with porcine intrinsic factor. The results of this test confirmed that the partially purified porcine R protein sample contained porcine TCN1 and could be used to generate anti-porcine TCN1 monoclonal antibodies. [Example]

[0139] Generation of anti-porcine TCN1 monoclonal antibodies using crude preparations of porcine R protein from natural sources In this example, a sample of native porcine R protein partially purified from a powdered crude extract of porcine gastric mucosa with vitamin B12-Sepharose (Figure 1) was used to immunize five BALB / c and ten A / J female mice to generate monoclonal antibodies against porcine R protein. Mice received three intraperitoneal injections of the immunogen (50 μg / dose / animal) at three-week intervals. The primary immunization used antigen emulsified in complete Freund's adjuvant (CFA), followed by subsequent booster immunizations in incomplete Freund's adjuvant (IFA).

[0140] One week after the final injection, mice were bled, and serum samples were tested in an ELISA assay for binding to porcine TCN1-specific antibodies and synthetic peptides. The 171B 1G5 mAb was used as a positive control for the presence of porcine TCN1 protein on coated ELISA plates (not shown). All animals immunized with partially purified porcine R proteins developed high antibody titers (>1:100,000) against the antigen. Figure 6 shows examples of Balb / C (Panel A) and A / J (Panel B) mouse bleed titers with different antigens. As can be seen, both strains of mice developed high antibody titers against porcine TCN1, but no binding was observed with the synthetic R1, R2, and R3 peptides. These results indicate that these peptides may not represent immunodominant epitopes of the native glycosylated porcine R protein.

[0141] Mice received prefusion boosts with the same immunogen (10 μg / dose / animal in PBS, IP) on three consecutive days, and mouse spleens were harvested and frozen on day 4. Several years later, when recombinant piglet TCN1 protein becomes commercially available, thawed immune splenocytes will be used for hybridoma preparation according to the standard procedures described above in Example 1. Hybridoma supernatants were screened for binding to both rec piglet TCN1 (LifeSpan BioSciences, LS-G23154, Seattle, WA) and partially purified native pig R protein adsorbed to ELISA plates at 1 μg / mL in a volume of 50 μL per well. Several hybridomas giving positive signals with both antigens were selected. As shown in Figure 7, the monoclonal antibodies produced by the 171J 3F1 and 171J 3A6 clones bound rec piglet TCN1 much better than native piglet R proteins; monoclonal antibody 171J 9G7 recognized both antigens equally well, and 171J 5H12 mAb bound native piglet R proteins more strongly than rec piglet TCN1. No cross-reactivity between the monoclonal antibodies and porcine intrinsic factor was observed. Hybridomas were subcloned by limiting dilution, expanded, and frozen. Monoclonal antibodies were purified from hybridoma supernatants on a column of Protein A Sepharose (GE Healthcare, Chicago, IL).

[0142] Monoclonal antibody epitope mapping was performed on microarrays of linear 15-mer porcine TCN (UniProt ID: P17630.2) and porcine intrinsic factor (UniProt ID: F1RI90) peptides with a 14-amino acid peptide-peptide overlap by PEPperPRINT GmbH (Heidelberg, Germany). Briefly, peptide microarrays were incubated with 171B 1G5, 171J 3F1, 171J 3A6, 171J 5H12, or 171J 9G7 monoclonal antibodies at concentrations of 1, 10, and 100 μg / mL, then stained with secondary goat anti-mouse IgG (H+L) DyLight800 antibody and scanned using a LI-COR Odyssey Imaging System. Microarray image analysis and peptide annotation were performed using a PepSlide® Analyzer. Pre-staining of each peptide microarray variant with a secondary antibody did not reveal any background interactions that could interfere with the main assay.

[0143] Although none of the mAbs showed a response to linear porcine intrinsic factor peptides, even at antibody concentrations as high as 100 μg / ml, we successfully identified unique linear porcine TCN1 epitopes for the 171B 1G5, 171J 3F1, 171J 3A6, and 171J 5H12 antibodies. No epitopes were shared by more than two antibodies, and analysis of their amino acid sequences with FASTA software (EMBL-EBI, Hinxton, Cambridge) confirmed that all identified epitopes were unique to porcine TCN1. Figure 8 shows an overview of the epitope mapping of mouse mAbs assayed against the porcine TCN1 peptide microarray.

[0144] Monoclonal antibody 171J 9G7 showed no response to linear porcine TCN1 or porcine intrinsic factor peptides, even at antibody concentrations as high as 100 μg / ml. This negative result may be due to the conformational or more complex discontinuous nature of the epitope, which cannot be mimicked by linear peptides.

[0145] The characteristics of the TCN1-specific monoclonal antibodies are shown in Table 4.

[0146] [Table 4]

[0147] The locations of the linear epitopes for the 171B 1G5, 171J 3F1, 171J 3A6, and 171J 5H12 monoclonal antibodies on the surface of the TCN1 molecule were determined as described above in Example 1. These locations are shown in Figure 9 in front (Panel A) and top (Panel B) views of the model crystal structure. As can be seen, the epitopes of all four mAbs are located on the same side of the protein globule, and therefore, can be seen in the sandwich assay of native porcine TCN1. This makes it difficult to currently use any mAb pair for detection. This conclusion was confirmed by ELISA and Biacore pairing experiments (data not shown).

[0148] Binding parameters of the interaction between anti-porcine TCN1 monoclonal antibody and TCN1 protein were determined in kinetic experiments using a Biacore T200 equipped with Biacore T200 Control Software Version 2.0.1 (GE Healthcare Bio-Sciences, Pittsburgh, PA). Anti-porcine TCN1 mAb was immobilized using amino coupling chemistry at 700–5,000 RU densities onto a carboxymethyl dextran surface on flow cells 2, 3, and 4 of a CM5 sensor chip. A control antibody was immobilized on flow cell 1 to subtract bulk effects. Immobilized mAb to recombinant piglet TCN1 (LifeSpan BioSciences, LS-G23154, Seattle, WA) or native pig R protein was injected onto the surface at a flow rate of 30 μL / min for 8 min, followed by 20 min of dissociation. Buffer blanks were run in triplicate with injections of running buffer (10 mM HEPES, 150 mM NaCl, 0.05% P-20, pH 7.4) and used to double-reference mAb binding data before fitting. After each binding cycle, the surface was regenerated with two sequential injections of 10 mM glycine, pH 2.0, and 0.1 M sodium bicarbonate, pH 8.5, for 30 seconds each, at a flow rate of 30 μL / min. Kinetic data were collected over antigen concentrations ranging from 25 to 1600 nM and globally fitted to a simple 1:1 interaction model. Binding parameters for porcine TCN1-specific mAb interactions were determined using Biacore T200 Evaluation Software Version 3.0 (GE Healthcare Bio-Sciences). Kinetic parameters for binding of anti-porcine TCN1 monoclonal antibodies to rec pig TCN1 and native pig R protein are shown in Table 5. As can be seen, three of the five antibodies interacted exclusively with recombinant piglet TCN1, and only the 171J 5H12 mAb bound native porcine R protein with extremely high affinity, forming a highly stable complex with this antigen. Monoclonal antibody 171J 9G7 recognized both antigens, but the affinity of the antibody for the recombinant protein was more than 500-fold higher than for the native protein.

[0149] These results can be explained by differences in glycosylation of the two antigens. The molecular weight of 46.3 kDa of the yeast-produced recombinant piglet TCN1 (LifeSpan BioSciences, LS-G23154, Seattle, WA) corresponds to the molecular weight of the non-glycosylated polypeptide chain. As previously mentioned, the native porcine R protein is heavily glycosylated, containing up to 50% w / w carbohydrate. Glycosylation, on the other hand, is important for the 171J This prevents the 3F1, 171J 3A6, and 171B 1G5 mAbs from binding to the native antigen and reduces the effectiveness of the interaction of the 171J 9G7 mAb with glycosylated porcine R protein. On the other hand, glycosylation promotes the binding of the 171J 5H12 mAb to native porcine TCN1, and the carbohydrate moiety is an important part of the epitope recognized by this antibody. This conclusion was confirmed by experiments using enzymatic deglycosylation of the native porcine R protein (data not shown).

[0150] [Table 5] [Example]

[0151] Sequencing of anti-porcine TCN1 monoclonal antibody Hybridoma cells produced as described in Examples 1 and 2, which produce monoclonal antibodies 171B 1G5, 171J 3F1, 171J 3A6, 171J 5H12, and 171J 9G7, were obtained, and total RNA was isolated therefrom according to the technical manual for the TRIZOL® Reagent (Thermo Fisher Scientific, Waltham, MA). The total RNA was then reverse transcribed into cDNA using either an isotype-specific antisense primer or a universal primer according to the technical manual for the PRIMESCRIPT™ First Strand cDNA Synthesis Kit (Takara Bio USA, Inc., Mountain View, CA). H , V L , CH , and C L The antibody fragments were amplified according to the standard operating procedure (SOP) for rapid amplification of cDNA ends (RACE) in GenScript (Piscataway, NJ). The amplified antibody fragments were separately cloned into standard cloning vectors. Colony PCR was performed to screen for clones with inserts of the correct size. Five or more colonies with inserts of the correct size were sequenced for each fragment. Each clone had >99% sequence identity to other clones. The sequences of different clones were aligned, and a consensus sequence was obtained.

[0152] Each DNA and amino acid sequence obtained for each monoclonal antibody was assigned a sequence identifier as outlined in Table 6. These sequences included the DNA sequence of both the heavy and light chains of each monoclonal antibody, the amino acid sequences of the heavy and light chains, the amino acid sequences of the heavy and light chain variable regions, and the amino acid sequences of the three complementarity determining regions (CDR1, CDR2, and CDR3) of both the heavy and light chain variable regions.

[0153] [Table 6]

[0154] Additionally, IMGT® analysis (ImMuno GeneTics Information System, Montpellier, France) of the V(D)J junctions of the heavy and light chain variable region sequences of each monoclonal antibody is shown in Table 7.

[0155] [Table 7]

[0156] The isotype of monoclonal antibody 171B 1G5 was mouse IgG2b, kappa.

[0157] Using the sequence information of the anti-porcine TCN1 monoclonal antibody, synthetic heavy and light chain genes were synthesized and transiently expressed in mammalian cells to produce small amounts of recombinant antibody. The expressed antibody was purified on a protein A affinity column and analyzed by ELISA for porcine TCN1 binding as described above. The antibodies were tested in a ELISA assay. Figure 10 shows exemplary ELISA titration results for recombinant piglet TCN1 using conventionally produced 171B 1G5 monoclonal antibody versus recombinantly produced 171B 1G5 monoclonal antibody. As can be seen, the data in Figure 10 confirms that the sequence obtained for the antibody is accurate, as the two titration curves are superimposable. [Example]

[0158] Purification of native porcine TCN1 protein from crude preparations on an affinity column using 171J 5H12 monoclonal antibody In this example, monoclonal antibody 171J 5H12 was used to prepare an affinity sorbent for purifying native porcine R protein from crude preparations. The affinity resin was prepared by covalently immobilizing 30 mg of anti-porcine TCN1 monoclonal antibody 171J 5H12 to 3 g of CNBr-Sepharose 4B Fast Flow (GE Healthcare Bio-Sciences, #17-0981-01, Pittsburgh, PA) using the manufacturer's protocol and resuspended in PBS, pH 7.4. A 20% solution of crude powdered extract of porcine gastric mucosa was prepared by suspending 42 g of powder in 220 mL of distilled water containing 0.02% sodium azide and stirring at 40°C for 16 hours. The resulting suspension was centrifuged at 20,000 xg for 1 hour at 40°C, and the cloudy supernatant was decanted from the pellet, transferred to a new tube, and centrifuged again at 20,000 xg for 40 minutes at 40°C.

[0159] Clarified aqueous extract of crude porcine gastric mucosa (approximately 180 mL) was mixed with 171J 5H12 mAb-Sepharose (approximately 10 mL) and gently agitated overnight at room temperature on an orbital shaker. The affinity resin was separated from the supernatant by vacuum aspiration through a Buchner funnel containing a medium-sized sintered glass disk, washed three times with PBS, pH 7.4, and packed into a glass column (1.5 x 7.2 cm, V = 12.7 mL). The column was washed with 100 mL PBS, pH 7.4, followed by 100 mL of 0.1 M sodium citrate, pH 5.0, at a flow rate of 10 mL / min. Bound protein was eluted from the column with 100 mL of 0.1 M sodium citrate, pH 2.2, and fractions were monitored by absorbance at 280 nm. Protein-containing fractions were pooled and dialyzed against two changes (2 L each) of PBS containing 0.02% sodium azide, pH 7.4. A total of 7.8 mg of protein was purified from 42 g crude powder extract of porcine gastric mucosa.

[0160] As shown in Figure 11, like native porcine TCN1, the protein purified on an affinity column using the 171J 5H12 mAb migrates as a single broad band of 70–92 kDa on SDS-PAGE under reducing conditions (Panel A) and has a highly acidic isoelectric point (pI < 4.5, Panel B).

[0161] Protein identification was performed by Bio-Synthesys (Lewisville, TX) using trypsin digestion of 70-92 kDa proteins from gel slices. Peptides were analyzed by nanocapillary LC-MS / MS followed by data mining. The predominant protein in the sample was identified as porcine transcobalamin I, and the peptides represented 67% of the porcine TCN1 sequence.

[0162] This example demonstrates that monoclonal antibody 171J 5H12 can be used to prepare an affinity resin that can be used to isolate homogeneous native porcine TCN1 in a single step from crude extracts of porcine gastric mucosa under extremely mild conditions. [Example]

[0163] Detection of native porcine TCN1 using ELISA with 171J 5H12 mAb In this example, monoclonal antibody 171J 5H12 was used in an ELISA assay to detect native porcine R protein in crude preparations and purified samples of porcine intrinsic factor. A standard titration curve was generated using column-purified native porcine TCN1 affinity (Figure 11) using immobilized 171J 5H12 monoclonal antibody as the standard. All ELISA steps were performed at room temperature. Nunc Maxi-Sorp™ flat-bottom ELISA plates were coated with 2 μg / mL vitamin B12-BSA conjugate in PBS at 50 μL per well for 1 hour. The plates were tapped dry, blocked with 200 μL of blocking solution per well, and washed as described above. Two-fold dilutions of native porcine TCN1 (2000 ng / mL) and samples to be tested were made in PBS and added to the plate wells at a volume of 50 μL per well. After 1 hour of incubation and washing of the plate, 171J 5H12 mAb was added at 1 μg / mL in PBS at 50 μL per well. The plate was incubated for 1 hour, washed, and then goat anti-mouse IgG-HRP conjugate diluted 1:3,000 in blocking solution was added at 50 μL per well for 1 hour. The plate was washed again, and TMB substrate (Moss, Pasadena, MD) was added at 100 μL per well for 15 minutes. The plate was read at 650 nm using an ELISA plate reader.

[0164] Figure 12 demonstrates the detection of native porcine TCN1 in crude extracts of porcine gastric mucosa using an ELISA assay with monoclonal antibody 171J 5H12. As can be seen, the assay allows for the detection of at least 1 ng / mL of native porcine TCN1 in the sample. [Example]

[0165] Porcine intrinsic factor sample polishing with affinity resin using 171J 5H12 mAb In this example, the monoclonal antibody 171J 5H12, specific for native porcine TCN1, was used to develop affinity resins for use in removing porcine TCN1 contaminants from porcine intrinsic factor preparations and / or, if necessary, polishing purified HIF. Samples of porcine intrinsic factor were tested using the ELISA assay described in Example 5, which detected low levels of porcine TCN1 contamination. To remove contaminants, samples were depleted with affinity resins using immobilized 171J 5H12 mAb. 100 μL of 171J 5H12-Sepharose 4B, prepared as described in Example 4, was added to 300 μL of HIF (1 mg / mL) and incubated for 5 hours at room temperature with intermittent gentle mixing. The affinity resin was removed by centrifugation at 3000 g for 15 minutes at room temperature, and the supernatant was tested for the presence of native porcine TCN1 in an ELISA assay. Figure 13 shows the results of polishing a porcine intrinsic factor sample with 171J 5H12 mAb-Sepharose. As can be seen, after depletion the level of porcine TCN1 contamination decreased from 2.7% to 0.1% of total protein.

[0166] Thus, there have been provided in accordance with the present disclosure compositions and methods of making and using the same that fully satisfy the objects and advantages set forth above. While the present disclosure has been described in conjunction with the specific figures, experiments, results, and language set forth above, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the present disclosure.

Claims

1. 1. A method for detecting TCN1 present in a porcine intrinsic factor (HIF) preparation, comprising: contacting the HIF preparation with an antibody or antigen-binding fragment thereof capable of specifically binding to an epitope of porcine transcobalamin-1 (TCN1) comprising SEQ ID NO: 3 or 23 under conditions such that an antibody / TCN1 complex is formed if porcine transcobalamin-1 (TCN1) is present in the HIF preparation; detecting any antibody / TCN1 complexes formed, wherein the amount of antibody-TCN1 complex formed is directly proportional to the amount of TCN1 present in the HIF preparation; The method comprising:

2. The method of claim 1, wherein the antibody or antigen-binding fragment thereof is attached to a label used in detecting the antibody / TCN1 complex.

3. 1. A method for removing TCN1 present in a porcine intrinsic factor (HIF) preparation, comprising: contacting the HIF preparation with an antibody or antigen-binding fragment thereof that specifically binds to an epitope of TCN1 comprising SEQ ID NO: 3 or 23 under conditions such that an antibody / TCN1 complex is formed if TCN1 is present in the HIF preparation; removing the antibody / TCN1 complex from the HIF preparation; The method comprising:

4. 4. The method of claim 3, wherein the antibody or antigen-binding fragment thereof is attached to a solid support that aids in the removal of the antibody-TCN1 complex from the HIF preparation.

5. 4. The method of claim 3, further comprising the step of eluting TCN1 from the antibody / TCN1 complex, thereby purifying TCN1 from the HIF preparation.

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