Anti-thyroid hormone (T4) recombinant antibody or antigen-binding fragment

Abstract

The present invention relates to the field of antibodies, antigen-binding fragments and immunodiagnosis. Specifically, the present invention relates to recombinant antibodies or antigen-binding fragments for binding thyroid hormone T4 (thyroxine) and halogenated bisphenol A. Furthermore, the present invention relates to isolated nucleic acid molecules comprising nucleotide sequences encoding the recombinant antibodies or antigen-binding fragments of the present invention, as well as expression vectors and host cells comprising the nucleic acid molecules of the present invention. In addition, the present invention relates to methods for producing recombinant antibodies or antigen-binding fragments for binding T4 thyroid hormone and halogenated bisphenol A, test kits and immunoassays comprising the recombinant antibodies or antigen-binding fragments of the present invention, and methods for determining the levels of T4 thyroid hormone and / or halogenated bisphenol A in a sample of a subject. Furthermore, the present invention relates to methods for processing samples, such as immunoaffinity-based sample preparation methods for enriching halogenated bisphenol A from a sample.

Description

Field of the Invention

[0001] The present invention relates to the field of antibodies, antigen-binding fragments and immunodiagnosis. In particular, the present invention relates to recombinant antibodies or antigen-binding fragments for binding thyroid hormone T4 (thyroxine) and halogenated bisphenol A. Furthermore, the present invention relates to isolated nucleic acid molecules comprising nucleotide sequences encoding the recombinant antibodies or antigen-binding fragments of the present invention, and expression vectors and host cells comprising the nucleic acid molecules of the present invention. In addition, the present invention relates to methods for producing recombinant antibodies or antigen-binding fragments for binding T4 thyroid hormone and halogenated bisphenol A, test kits and immunoassays comprising the recombinant antibodies or antigen-binding fragments of the present invention, and methods for determining the levels of T4 thyroid hormone and / or halogenated bisphenol A in a sample of a subject. Furthermore, the present invention relates to methods for processing samples, such as immunoaffinity-based sample preparation methods for enriching halogenated bisphenol A from a sample. Background Art

[0002] Thyroid hormones play an important role in differentiation, growth and metabolism. The thyroid hormones, triiodothyronine (T3) and thyroxine (T4) are produced by the thyroid gland. The precursor of T4 is the tyrosine amino acid of thyroglobulin expressed in the thyroid gland. The tyrosine amino acid is bound to iodine, two iodinated tyrosines are conjugated and subsequently cleaved from thyroglobulin. T4 is further converted to T3 by deiodinases within the target cells. The difference between T4-hormone and T3-hormone is only one iodine atom.

[0003] T4 is the dominant thyroid hormone in serum, with a concentration about 40 times higher than that of T3 (90 nM and 2 nM, respectively). Only 0.03% of the total T4 (10-25 pM) and 0.3% of T3 (4.0-8.0 pM) in serum enter cells in an unbound form and are biologically active (Yen, PMPhysiological Reviews (2001) 81: 1097-1142).

[0004] Currently, the common test used to analyze T4 from serum is the radioimmunoassay (RIA). The determination of free T4 is an important means for screening for potential thyroid dysfunction. When abnormal values ​​are obtained, a more thorough diagnosis is performed to clarify the cause of the thyroid dysfunction (free and bound T4, T3, TSH and autoantibodies). In some diseases, increased amounts of T3 are produced, while T4 levels are normal. Therefore, it is important to perform multiple diagnostics to fully understand thyroid function.

[0005] T4 has one more iodine than T3. This small difference between the most important thyroid hormones makes it very challenging to find specific antibodies against T3 or T4. In addition, both thyroid hormones T3 and T4 are relatively small and hydrophobic.

[0006] Some commercial antibody products, whether polyclonal or monoclonal, are available for thyroid hormones. In addition, some patent applications relate to thyroid hormone antibodies. For example, JP2010178649 (A) describes an anti-thyroxine antibody comprising a specific heavy chain variable region (V H ) and specific light chain variable region (V L Islam et al. (2011) described an antibody for T4 in a recombinant Fab format that was selected using phage display technology and used in a one-step open immunoassay (Islam KN et al. Analytical Chemistry (2011) 83: 1008-1014).

[0007] Furthermore, in order to determine the thyroid hormone T4 in a biological sample, an antibody or antigen-binding fragment having a particularly high affinity for T4 is required. Summary of the invention

[0008] The present invention relates to a novel reagent for rapid and reliable detection of T4 thyroid hormone and a novel reagent for rapid and reliable detection of halogenated bisphenol A.

[0009] The present invention discloses surprising binding properties of antibodies or antigen-binding fragments capable of binding thyroid hormone and halogenated bisphenol A. In fact, the present invention relates to recombinant antibodies or antigen-binding fragments capable of binding thyroid hormone T4 with high affinity and capable of halogen-dependent binding to halogenated bisphenol A.

[0010] A surprising advantage of the present invention includes that the levels of halogenated bisphenol A observed in humans do not interfere with immunoassays when using high affinity anti-T4 antibodies. However, the cross-reactivity of the T4 thyroid hormone binding antibodies or antigen-binding fragments of the present invention to halogenated bisphenol A allows the same antibody to be used for measurement and / or sample preparation of halogenated bisphenol A from, for example, biological samples, environmental samples, or food samples. The cross-reactivity also provides an effective means for evaluating the endocrine disruptor potential of organic molecules that have an effect on the thyroid hormone system.

[0011] The present invention relates to recombinant antibodies or antigen-binding fragments (eg, isolated antibodies or antigen-binding fragments) for binding T4 thyroid hormone and halogenated bisphenol A.

[0012] Furthermore, the present invention relates to an isolated (recombinant) nucleic acid molecule comprising a nucleotide sequence encoding a recombinant antibody or antigen-binding fragment of the present invention.

[0013] Furthermore, the present invention relates to an expression vector comprising the nucleic acid molecule of the present invention.

[0014] Furthermore, the present invention relates to a host cell comprising the nucleic acid molecule or expression vector of the present invention.

[0015] In addition, the present invention relates to a method for producing the recombinant antibody or antigen-binding fragment of the present invention for binding T4 thyroid hormone and halogenated bisphenol A (such as triiodobisphenol or tetrahalogenated bisphenol A), wherein the method comprises introducing the expression vector of the present invention into a host cell and growing the cell under conditions that allow the production of the antibody or antigen-binding fragment.

[0016] In addition, the present invention relates to a test kit comprising the recombinant antibody or antigen-binding fragment of the present invention. Furthermore, the use of the test kit for detecting T4 thyroid hormone and / or halogenated bisphenol A in a sample is within the scope of the present invention. In addition, the use of the kit comprising the recombinant antibody or antigen-binding fragment of the present invention for processing a sample is within the scope of the present invention.

[0017] Still further, the present invention relates to a method for determining one or more concentrations of T4 thyroid hormone and / or halogenated bisphenol A in a sample, wherein the method comprises allowing the recombinant antibody or antigen-binding fragment of the present invention to contact a sample (such as a biological, environmental or food sample, or a sample from a subject), and thereafter determining the concentrations of T4 thyroid hormone and / or halogenated bisphenol A in the sample, respectively.

[0018] Still further, the present invention relates to a method for treating (e.g., pretreating) a sample, wherein the method comprises allowing the recombinant antibody or antigen-binding fragment of the present invention to contact a sample (such as a biological sample, an environmental sample, a material sample or a food sample, or a sample from a subject).

[0019] And furthermore, the present invention relates to immunoassays comprising the recombinant antibodies or antigen-binding fragments of the present invention.

[0020] And still further, the present invention relates to the use of the recombinant antibody or antigen-binding fragment or immunoassay of the present invention for screening for endocrine disruptor potential present in a sample, such as endocrine disruptor potential associated with the thyroid axis.

[0021] Other objects, details and advantages of the present invention will become apparent from the following drawings, detailed description and examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 a shows the nucleotide sequence of the T4 Fab light chain (SEQ ID NO: 1 or SEQ ID NO: 7 (in SEQ ID NO: 7, each DNA codon NNN is independently selected from the group consisting of DNA codons TAC, TAT, TTT and TTC)).

[0023] FIG1 b shows the nucleotide sequence of the T4 Fab heavy chain (SEQ ID NO: 2 or SEQ ID NO: 8 (in SEQ ID NO: 8, each DNA codon NNN is independently selected from the group consisting of DNA codons TAC, TAT, TTT and TTC)).

[0024] Figure 2a shows the amino acid sequence of the T4 Fab light chain (SEQ ID NO: 3 or SEQ ID NO: 5 (in SEQ ID NO: 5, each X is independently Y (tyrosine) or F (phenylalanine)). The CDR regions are marked in bold. The three complementary regions LCDR1 (amino acids 24-33 of SEQ ID NO: 3 or SEQ ID NO: 5, according to Kabat amino acids 24-34), LCDR2 (amino acids 49-55 of SEQ ID NO: 3 or SEQ ID NO: 5, according to Kabat amino acids 50-56) and LCDR3 (amino acids 88-96 of SEQ ID NO: 3 or SEQ ID NO: 5, according to Kabat 89-97) of each immunoglobulin chain are shown.

[0025] Figure 2b shows the amino acid sequence of the T4 Fab heavy chain (SEQ ID NO:4 or SEQ ID NO:6 (in SEQ ID NO:6, each X is independently Y (tyrosine) or F (phenylalanine)). The CDR regions are marked in bold. The three complementary regions of each immunoglobulin chain are shown: HCDR1 (amino acids 31-35 of SEQ ID NO:4 or SEQ ID NO:6, according to Kabat amino acids 31-35), HCDR2 (amino acids 50-66 of SEQ ID NO:4 or SEQ ID NO:6, according to Kabat amino acids 50-65) and HCDR3 (amino acids 99-113 of SEQ ID NO:4 or SEQ ID NO:6, according to Kabat amino acids 95-102).

[0026] Figure 3a shows the amino acid sequence of the T4 Fab light chain (according to SEQ ID NO: 3). The CDR regions are underlined and the amino acids responsible for T4 binding are identified (at least Y93).

[0027] Figure 3b shows the amino acid sequence of the T4 Fab heavy chain (according to SEQ ID NO: 4). The CDR regions are underlined, and the amino acids responsible for T4 binding are identified (at least Y33, Y107, Y109 and / or M111, or any combination thereof).

[0028] Figure 4 shows the affinity and specificity data of T4 Fab determined by surface plasmon resonance (SPR).T4-alkaline phosphatase was immobilized on the sensor surface and the Kd value was determined by inhibition assay.

[0029] Figure 5 The results of ELISA assay for L-thyroxine are revealed.

[0030] Figure 6 The results of ELISA assay for tetraiodobisphenol A are revealed.

[0031] Figure 7 The results of ELISA assay for tetrabromobisphenol A are disclosed.

[0032] Figure 8 The results of ELISA assay for tetrachlorobisphenol A are disclosed.

[0033] Fig. 9 The results of ELISA assay for bisphenol A are revealed.

[0034] Figure 10a reveals that the interatomic distances between atoms of T4 and TIBPA ligand and anti-T4 Fab are up to

[0035] Figure 10b reveals the interatomic distances responsible for halogen bonding. Figure 10b_1 shows the relevant halogen bonds in the structures of anti-T4 Fab and T4. A) I5'···CD2 distance is And the I5'···CG distance is Residue Y107 of the T4Fab heavy chain is involved. The I3'···SD distance is The C3'-I3'···SD angle is 158.31°, involving residue M111 of the T4 Fab heavy chain; B) the I5'···CE2-CD2 bond center distance is The C5'-I5'···CE2-CD2 bond center angle is 166.37°, involving residue Y107 of the T4 Fab heavy chain; C) the I5'···Y107 ring center distance is The C5'-I5'···Y107 ring center angle is 173.54°. Figure 10b_2 shows the relevant halogen bonds in the structure of anti-T4Fab and tetraiodobisphenol A. A) The I4'···CD2 distance is And the I4'···CG distance is Residue Y107 of the T4Fab heavy chain is involved; the I3'···SD distance is The C2-I3'···SD angle is 153.05°, involving residue M111. The I2···O distance is The C15-I2···O angle is 148.38°, involving the backbone oxygen of residue S57 of the T4Fab heavy chain; B) the I4'···CD2-CG bond center distance is The C4-I4'···CD2-CG bond center angle is 152.91°, involving residue Y107 of the T4 Fab heavy chain; C) the I4'···Y107 ring center distance is And the center angle of C4-I4'···Y107 is 169.54°.

[0036] Figure 11a shows the variable domains of the T4 Fab fragment of the crystal structure. The L-chain is dark grey and the H-chain is light grey. The bound T4 molecule is shown as a stick model. The six CDR loops (L1, L2, L3, H1, H2 and H3) of the Fab fragment are labeled.

[0037] Figure 11b shows the binding groove of the T4 Fab fragment and the bound T4 molecule. The residues involved in T4 binding are shown as stick models and are labeled.

[0038] Sequence Listing

[0039] Sequences or partial sequences of anti-T4 thyroid hormone antibodies (eg, T4) or antigen-binding fragments of the present invention

[0040] SEQ ID NO: 1: T4 Fab light chain nucleotide sequence

[0041] SEQ ID NO:2: T4 Fab heavy chain nucleotide sequence

[0042] SEQ ID NO:3:T4 Fab light chain amino acid sequence

[0043] SEQ ID NO:4: T4 Fab heavy chain amino acid sequence

[0044] SEQ ID NO:5: T4 Fab light chain amino acid sequence (wherein each X is independently Y or F)

[0045] SEQ ID NO:6: T4 Fab heavy chain amino acid sequence (wherein each X is independently Y or F)

[0046] SEQ ID NO:7: T4 Fab light chain nucleotide sequence (wherein each DNA codon nnn is independently selected from the group consisting of DNA codons TAC, TAT, TTT and TTC)

[0047] SEQ ID NO:8: T4 Fab heavy chain nucleotide sequence (wherein each DNA codon nnn is independently selected from the group consisting of DNA codons TAC, TAT, TTT and TTC) DETAILED DESCRIPTION

[0048] In one embodiment of the invention, the recombinant antibody or antigen-binding fragment of the invention binds (eg, specifically) to T4 thyroid hormone.

[0049] Thyroxine (3,5,3',5'-tetraiodothyronine) or T4 thyroid hormone belongs to the group of thyroid hormones (T 4 and T 3 ) and is produced by follicular cells of the thyroid gland. 4 Convert to T 3 When T 4 The role is through T 3 Mediated.

[0050] T4 is the most commonly measured thyroid hormone for diagnosing thyroid function. Thyroid function is studied by detecting T4 levels in blood samples. T4 mainly affects protein synthesis and oxygen consumption in all tissues, but it is also important for growth, development and sexual maturity. Increased T4 levels have been found in samples of patients who, for example, suffer from hyperthyroidism due to Grave's disease or Plummer's disease or acute or subacute thyroiditis. Low levels of T4 have been associated with congenital hypothyroidism, myxedema, chronic thyroiditis and some chronic genetic abnormalities.

[0051] T4 can be measured as free T4, which is an indicator of T4 activity in the body. The free T4 index is related to total T4 multiplied by thyroid hormone intake, which in turn is a measure of unbound T4 binding globulin. In one embodiment of the invention, T4 is measured as free T4, for example by using a sensor. T4 can also be measured as total T4, which depends on T4 bound to T4 binding globulin. In one embodiment of the invention, T4 is measured as total T4, for example by using an immunoassay (such as ELISA or RIA) or a sensor or any suitable method known to those skilled in the art.

[0052] In one embodiment of the present invention, the recombinant antibody or antigen-binding fragment of the present invention binds (eg, specifically) to halogenated bisphenol A.

[0053] Bisphenol A and its halogenated analogs are commonly used industrial chemicals with strong toxicological effects in many organisms. BPA is used to make certain plastics and epoxy resins. Bisphenol A is an organic synthetic compound with the chemical formula (CH 3 ) 2 C(C 6 H 4 OH) 2, belongs to the group of diphenylmethane derivatives and bisphenols, has two hydroxyphenyl groups. It is a colorless solid that is soluble in organic solvents but poorly soluble in water.

[0054] Bisphenol A has the following formula I:

[0055]

[0056] As used herein, "halogenated bisphenol A" refers to bisphenol A that has been halogenated with 1 or more halogens (e.g., one, two, three, four, five or six halogens, such as 1-4 halogen substituents). In very specific embodiments, the halogenated bisphenol A is triiodobisphenol A or tetrahalogen bisphenol A. The one or more halogens in the halogenated bisphenol A are independently selected from the group consisting of fluorine (F), chlorine (Cl), bromine (Br) and iodine (I). In very specific embodiments, bisphenol A is halogenated with iodine, bromine, chlorine or a combination thereof. In other very specific embodiments of the present invention, the halogenated bisphenol A includes 1, 2, 3 or 4 iodine, bromine or chlorine substituents.

[0057] In one embodiment, the bisphenol A derivative has the following formula II:

[0058]

[0059] Wherein bisphenol A has been in one or more positions X 1-4 The halogenated halogenated moiety is halogenated with one or more substituents (i.e., one, two, three, or four halogen substituents), wherein each substituent is independently selected from the group consisting of fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0060] In one embodiment of the present invention, T4 thyroid hormone and halogenated bisphenol A are detected simultaneously.

[0061] As used herein, "antibody or antigen-binding fragment" refers to any antibody or fragment thereof that is capable of binding to T4 thyroid hormone. The term includes, for example, any fragment or single-chain antibody (e.g., Fab, scFv) that has the desired biological activity. For example, any complementary determining region, heavy chain variable region, light chain variable region, and any combination thereof are included within the scope of "antigen-binding fragment". "Fragment" as used in this context refers to any portion of a polypeptide or protein.

[0062] As used herein, "antibody" refers to a polypeptide or protein produced by an immune cell or by recombinant technology that responds to an antigen. An antibody is an immunoglobulin molecule, and it can belong to any of the classes IgG, IgM, IgE, IgA, or IgD; IgG and IgM are the most commonly used. As used herein, "binding polypeptide" refers to a polypeptide that binds to an antigen of interest. In some embodiments, the binding polypeptide may inhibit or suppress the function of the antigen of interest (polypeptide, protein, polysaccharide).

[0063] In antibodies, the variable loops between the β chains, each light (V L ) chain and heavy (V H ) chain, responsible for binding to the antigen. These loops are called complementarity determining regions (CDRs). As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody or its fragment that are responsible for antigen binding. The hypervariable region includes amino acid residues from CDR (e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain, and residues 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain, according to Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or other amino acid residues from "hypervariable loops" (framework regions). "Framework region" or "FR" residues are those variable domain residues except for the very specific hypervariable region residues defined by the number of residues in the present disclosure. As used herein, the terms "heavy chain variable domain", "V H Domain" and / or "V H ” are used interchangeably and refer to the hypervariable region of the heavy chain of an antibody (including both CDRs and framework domains); the terms “light chain variable domain”, “V L Domain" and / or "V L ” are used interchangeably and refer to the hypervariable region (including both CDRs and framework domains) of the heavy chain of an antibody.

[0064] At the molecular level, an antigen can be characterized by its ability to bind to the variable Fab region or antigen binding fragment of an antibody.As used herein, a "ligand" is a molecule that forms a complex with, for example, an anti-T4 antibody Fab or scFv.

[0065] "Fv" (variable domain) refers to the variable region of an immunoglobulin molecule that is responsible for ligand binding. "Single-chain Fv fragment (scFv)" refers to the V domains of an immunoglobulin molecule connected by a linker in a single polypeptide synthesized from a single mRNA molecule. H and V L Fragments of the variable domain.

[0066] In one embodiment of the invention, the recombinant antibody or antigen binding fragment comprises a constant region that corresponds to a human or non-human constant region. In other embodiments, at least a portion of the variable region of the recombinant antibody or antigen binding fragment corresponds to a human or non-human variable region.

[0067] As used herein, "Fab fragment" (fragment antigen binding) refers to a region of an antibody consisting of the variable and constant domains of an immunoglobulin light chain covalently linked by disulfide bridges to the variable and first constant domains of an immunoglobulin heavy chain.

[0068] In one embodiment of the invention, the recombinant antibody or antigen-binding fragment comprises one or more (e.g., at least two, at least three, at least four, at least five or at least six) complementary determining regions (CDRs) amino acids selected from the group consisting of:

[0069] light chain region 1, comprising amino acids corresponding to amino acids 24-33 of SEQ ID NO:3,

[0070] light chain region 2, comprising amino acids corresponding to amino acids 49-55 of SEQ ID NO:3,

[0071] light chain region 3, comprising amino acids corresponding to amino acids 88-96 of SEQ ID NO:3,

[0072] heavy chain region 1, comprising amino acids corresponding to amino acids 31-35 of SEQ ID NO:4,

[0073] Heavy chain region 2, including amino acids corresponding to amino acids 50-66 of SEQ ID NO: 4,

[0074] and

[0075] Heavy chain region 3, includes amino acids corresponding to amino acids 99-113 of SEQ ID NO:4.

[0076] In one embodiment of the invention, the recombinant antibody or antigen-binding fragment comprises one or more (e.g., at least two, at least three, at least four, at least five or at least six) complementary determining regions (CDRs) amino acids selected from the group consisting of:

[0077] light chain region 1, comprising amino acids corresponding to amino acids 24-33 of SEQ ID NO:5,

[0078] light chain region 2, comprising amino acids corresponding to amino acids 49-55 of SEQ ID NO:5,

[0079] light chain region 3, comprising amino acids corresponding to amino acids 88-96 of SEQ ID NO:5,

[0080] heavy chain region 1, comprising amino acids corresponding to amino acids 31-35 of SEQ ID NO:6,

[0081] Heavy chain region 2, including amino acids corresponding to amino acids 50-66 of SEQ ID NO: 6,

[0082] and

[0083] Heavy chain region 3, includes amino acids corresponding to amino acids 99-113 of SEQ ID NO:6.

[0084] In one embodiment of the invention, the recombinant antibody or antigen-binding fragment comprises a heavy chain variable domain amino acid sequence comprising a CDR3 as set forth in amino acids 99-113 of SEQ ID NO: 4. In one embodiment of the invention, the recombinant antibody or antigen-binding fragment comprises a heavy chain variable domain amino acid sequence comprising a CDR3 as set forth in amino acids 99-113 of SEQ ID NO: 6.

[0085] In one embodiment of the invention, the recombinant antibody or antigen-binding fragment comprises a heavy chain variable domain amino acid sequence comprising a CDR3 as set forth in amino acids 99-113 of SEQ ID NO: 4 and a CDR2 as set forth in amino acids 50-66 of SEQ ID NO: 4 or a CDR1 as set forth in amino acids 31-35 of SEQ ID NO: 4. In one embodiment of the invention, the recombinant antibody or antigen-binding fragment comprises a heavy chain variable domain amino acid sequence comprising a CDR3 as set forth in amino acids 99-113 of SEQ ID NO: 6 and a CDR2 as set forth in amino acids 50-66 of SEQ ID NO: 6 or a CDR1 as set forth in amino acids 31-35 of SEQ ID NO: 6.

[0086] In one embodiment of the invention, the recombinant antibody or antigen-binding fragment comprises a heavy chain variable domain amino acid sequence comprising a CDR1 as set forth in amino acids 31-35 of SEQ ID NO: 4, a CDR2 as set forth in amino acids 50-66 of SEQ ID NO: 4, and a CDR3 as set forth in amino acids 99-113 of SEQ ID NO: 4. In one embodiment of the invention, the recombinant antibody or antigen-binding fragment comprises a heavy chain variable domain amino acid sequence comprising a CDR1 as set forth in amino acids 31-35 of SEQ ID NO: 6, a CDR2 as set forth in amino acids 50-66 of SEQ ID NO: 6, and a CDR3 as set forth in amino acids 99-113 of SEQ ID NO: 6.

[0087] In one embodiment of the present invention, the recombinant antibody or antigen-binding fragment comprises:

[0088] a heavy chain variable domain amino acid sequence comprising a CDR1 as set forth in amino acids 31-35 of SEQ ID NO:4, a CDR2 as set forth in amino acids 50-66 of SEQ ID NO:4, and a CDR3 as set forth in amino acids 99-113 of SEQ ID NO:4; and

[0089] A light chain variable domain amino acid sequence containing CDR3 as set forth in amino acids 88-96 of SEQ ID NO:3.

[0090] In one embodiment of the present invention, the recombinant antibody or antigen-binding fragment comprises:

[0091] a heavy chain variable domain amino acid sequence comprising a CDR1 as set forth in amino acids 31-35 of SEQ ID NO:6, a CDR2 as set forth in amino acids 50-66 of SEQ ID NO:6, and a CDR3 as set forth in amino acids 99-113 of SEQ ID NO:6; and

[0092] A light chain variable domain amino acid sequence containing CDR3 as set forth in amino acids 88-96 of SEQ ID NO:5.

[0093] In one embodiment of the present invention, the recombinant antibody or antigen-binding fragment comprises:

[0094] a heavy chain variable domain amino acid sequence comprising a CDR1 as set forth in amino acids 31-35 of SEQ ID NO:4, a CDR2 as set forth in amino acids 50-66 of SEQ ID NO:4, and a CDR3 as set forth in amino acids 99-113 of SEQ ID NO:4; and

[0095] A light chain variable domain amino acid sequence containing CDR3 as set forth in amino acids 88-96 of SEQ ID NO:3 and CDR1 as set forth in amino acids 24-33 or CDR2 as set forth in amino acids 49-55 of SEQ ID NO:3.

[0096] In one embodiment of the present invention, the recombinant antibody or antigen-binding fragment comprises:

[0097] a heavy chain variable domain amino acid sequence comprising a CDR1 as set forth in amino acids 31-35 of SEQ ID NO:6, a CDR2 as set forth in amino acids 50-66 of SEQ ID NO:6, and a CDR3 as set forth in amino acids 99-113 of SEQ ID NO:6; and

[0098] A light chain variable domain amino acid sequence containing CDR3 as set forth in amino acids 88-96 of SEQ ID NO:5 and CDR1 as set forth in amino acids 24-33 or CDR2 as set forth in amino acids 49-55 of SEQ ID NO:5.

[0099] In a specific embodiment of the invention, the antibody or antigen-binding fragment comprises a light chain CDR 1-3 comprising or consisting of amino acids corresponding to amino acids 24-33, 49-55 and 88-96 of SEQ ID No: 3, respectively, and a heavy chain CDR 1-3 comprising or consisting of amino acids corresponding to amino acids 31-35, 50-66 and 99-113 of SEQ ID No: 4, respectively. In a specific embodiment of the invention, the antibody or antigen-binding fragment comprises a light chain CDR 1-3 comprising or consisting of amino acids corresponding to amino acids 24-33, 49-55 and 88-96 of SEQ ID No: 5, respectively, and a heavy chain CDR 1-3 comprising or consisting of amino acids corresponding to amino acids 31-35, 50-66 and 99-113 of SEQ ID No: 6, respectively.

[0100] In one embodiment of the present invention, the recombinant antibody or antigen-binding fragment comprises or consists of a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 3, and / or a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 4. In one embodiment of the present invention, the recombinant antibody or antigen-binding fragment comprises or consists of a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 5, and / or a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 6.

[0101] In one embodiment of the present invention, the heavy chain variable region (V H ) comprises or consists of amino acids 1-124 of SEQ ID NO: 4, and / or a light chain variable region (V L ) comprises or consists of amino acids 1-109 of SEQ ID NO: 3. In one embodiment of the present invention, the heavy chain variable region (V H ) comprises or consists of amino acids 1-124 of SEQ ID NO: 6, and / or a light chain variable region (V L ) comprises or consists of amino acids 1-109 of SEQ ID NO:5.

[0102] In one embodiment of the present invention, corresponding to Fig. 10A and / or Figure 3A -B, one or more amino acids of the recombinant antibody or antigen-binding fragment corresponding to the amino acid residues presented in SEQ ID NO: 3 and / or corresponding to the amino acids Y33, Y107, Y109 and M111 as defined in SEQ ID NO: 4 are responsible for binding to T4 (see, e.g. Figure 3A and B). In a specific embodiment, at least the amino acids of the recombinant antibody or antigen-binding fragment corresponding to amino acid Y93 of the light chain as defined in SEQ ID NO:3, and / or at least the amino acids of the recombinant antibody or antigen-binding fragment corresponding to amino acids Y33, Y107, Y109 and M111 of the heavy chain as defined in SEQ ID NO:4 bind to T4. Alternatively or in addition to any of the above Y93, Y33, Y107, Y109 and M111, amino acids of the recombinant antibody or antigen-binding fragment corresponding to, for example, D100, A50, N52, S57, Y59 and / or L47 of the heavy chain as defined in SEQ ID NO:4 and / or L95 of the light chain as defined in SEQ ID NO:3 may be responsible for binding to T4 and / or halogenated bisphenol A.

[0103] In one embodiment of the invention, at least one or more amino acids of the recombinant antibody or antigen-binding fragment corresponding to Y93 or F93 as defined in SEQ ID NO: 3 or SEQ ID NO: 5, and / or corresponding to amino acids Y33, F33, Y107, F107, Y109, F109 and M111 as defined in SEQ ID NO: 4 or SEQ ID NO: 6 are responsible for binding to T4 (see e.g. Fig. 10A , Figure 2A and B or Figure 3A and B). In a specific embodiment, the amino acids of the recombinant antibody or antigen-binding fragment corresponding to at least amino acids Y93 or F93 of the light chain as defined in SEQ ID NO:3 or SEQ ID NO:5, and / or the amino acids of the recombinant antibody or antigen-binding fragment corresponding to at least amino acids Y33, F33, Y107, F107, Y109, F109 and M111 of the heavy chain as defined in SEQ ID NO:4 or SEQ ID NO:6 are combined with T4. Alternatively or in addition to any of the above-mentioned Y93, F93, Y33, F33, Y107, F107, Y109, F109 and M111, the amino acids of the recombinant antibody or antigen-binding fragment corresponding to, for example, D100, A50, N52, S57, Y59, F59 and / or L47 of the heavy chain as defined in SEQ ID NO:4 or SEQ ID NO:6 and / or L95 of the light chain as defined in SEQ ID NO:3 or SEQ ID NO:5 may be responsible for binding to T4 and / or halogenated bisphenol A.

[0104] In one embodiment of the present invention, corresponding to Fig. 10A One or more amino acids of the recombinant antibody or antigen-binding fragment of one or more amino acid residues represented in or 10B are responsible for halogen binding. Fig. 10A Any tyrosine (Y) mentioned in can be replaced with phenylalanine (F) independently. In one embodiment, the amino acids of the recombinant antibody or antigen-binding fragment responsible for halogen binding are selected from the group consisting of: Y33, F33, Y107, F107, Y109, F109, M111, N52 and S57 corresponding to the amino acids defined in SEQ ID NO:4 or SEQ ID NO:6 and L95 and Y93 or F93 corresponding to the amino acids defined in SEQ ID NO:3 or SEQ ID NO:5. In a further embodiment of the invention, at least the amino acids corresponding to amino acids Y107 or F107 and M111 of the heavy chain defined in SEQ ID NO:4 or SEQ ID NO:6, or at least the amino acids corresponding to at least amino acids Y33 or F33, Y107 or F107, Y109 or F109 and M111 are responsible for halogen binding.

[0105] In other embodiments of the invention, the recombinant antibody or antigen-binding fragment comprises an amino acid sequence having 80-100% sequence identity to SEQ ID NO:3, 4, 5 or 6, for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, the antibodies or antigen-binding fragments according to the present invention include a heavy chain variable region and a light chain variable region, which include an amino acid sequence having 80-100% sequence identity to SEQ ID NO:3 and SEQ ID NO:4, or to SEQ ID NO:5 and SEQ ID NO:6, or to SEQ ID NO:3 and SEQ ID NO:6, or to SEQ ID NO:5 and SEQ ID NO:4, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In a further embodiment of the invention, the isolated nucleic acid molecule encoding the antibody or antigen-binding fragment of the invention comprises a polynucleotide sequence having 80-100% sequence identity to SEQ ID NO: 1, 2, 7 or 8, for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In a specific embodiment, the isolated nucleic acid molecule encoding the antibody or antigen-binding fragment of the invention comprises a polynucleotide sequence having 80-100% sequence identity to SEQ ID NOs: 1 and 2, or SEQ ID NOs: 7 and 8, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0106] In one embodiment of the present invention, the light chain of the recombinant antibody or antigen-binding fragment comprises an amino acid sequence having 80-100% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to amino acids 1-109 of SEQ ID NO:3 or SEQ ID NO:5, and / or the heavy chain of the recombinant antibody or antigen-binding fragment comprises an amino acid sequence having 80-100% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to amino acids 1-124 of SEQ ID NO:4 or SEQ ID NO:6.

[0107] The identity compared to any sequence of the disclosed sequence or its fragment refers to the identity compared to any sequence of the whole sequence of the present invention.As used herein, the % identity between two sequences is the function of the quantity of the same position shared by the sequences (i.e. % identity=same position # / position total # x 100), considering the number of rooms and the length of each room, these need to be introduced to carry out the best comparison of two sequences.The comparison of sequence and determination of the identity percentage between two sequences can be completed using existing mathematical algorithms in the prior art.This is applicable to both amino acid sequences and nucleotide sequences.For example, sequence identity can be determined by using BLAST (basic local alignment search tool) or FASTA (FAST-AII).In search, setting parameters "gap penalty" and "matrix" are typically selected as default values.

[0108] In one embodiment of the present invention, the recombinant antibody is a monoclonal antibody or the antigen binding fragment is a single-chain Fv or Fab fragment. In a specific embodiment, the antigen binding fragment is a single-chain Fv or Fab fragment comprising one or more CDRs selected from the group consisting of: light chain CDR1-3 containing amino acids 24-33, 49-55 and 88-96 of SEQ ID NO:3, respectively, and heavy chain CDR 1-3 containing amino acids 31-35, 50-66 and 99-113 of SEQ ID NO:4, respectively. In a specific embodiment, the antigen binding fragment is a single-chain Fv or Fab fragment comprising one or more CDRs selected from the group consisting of: light chain CDR 1-3 containing amino acids 24-33, 49-55 and 88-96 of SEQ ID NO:5, respectively, and heavy chain CDR 1-3 containing amino acids 31-35, 50-66 and 99-113 of SEQ ID NO:6, respectively. In a more specific embodiment, the single-chain Fv or Fab fragment comprises or consists of the amino acid sequence of SEQ ID NO: 3 and / or 4, SEQ ID NO: 5 and / or 6, SEQ ID NO: 3 and / or 6 or SEQ ID NO: 5 and / or 4.

[0109] In a very specific embodiment of the invention, the light chain of the antibody or antigen-binding fragment is a kappa (κ) type chain.

[0110] In a specific embodiment of the invention, the antibody or antigen-binding fragment is a fusion polypeptide, i.e., it has been fused (e.g., by genetic engineering) with, for example, any other fragment or polynucleotide to form a fusion polypeptide. Examples of suitable fusion partners include any conventional fusion partners known to those skilled in the art, such as, but not limited to, alkaline phosphatase, hydrophobic proteins, and any peptide suitable for functionalization.

[0111] In one embodiment of the present invention, the recombinant antibody or antigen-binding fragment binds to human or animal T4 thyroid hormone. In fact, the recombinant antibody or antigen-binding fragment is able to recognize the epitope of T4 thyroid hormone.

[0112] In one embodiment of the invention, the recombinant antibody or antigen-binding fragment has an affinity for T4 thyroid hormone of 800 pM or less (e.g., 10-800 pM or 30-600 pM or 200-530 pM, or 600 pM or less, or 530 pM or less) (e.g., when measured by competition ELISA and / or SPR sensor, e.g., as described in the article Tullila and Nevanen 2017, International Journal of Molecular Sciences 18(6), 1169). In other embodiments, the recombinant antibody or antigen-binding fragment has an affinity for halogenated bisphenol A of 800 nM or less (e.g., 10-800 nM, 30-600 nM, or 37-558 nM, or 600 nM or less, or 558 nM or less) (e.g., depending on the halogen (iodine, bromine, or chlorine)) (e.g., when measured by competition ELISA and / or SPR sensor, e.g., as described in the article Tullila and Nevanen, 2017, International Journal of Molecular Sciences 18(6), 1169). In a further embodiment of the invention, the recombinant antibody or antigen-binding fragment has an affinity for T4 thyroid hormone of 800 pM or less (e.g. 10-800 pM or 30-600 pM or 200-530 pM, or 600 pM or less, or 530 pM or less) and an affinity for halogenated bisphenol A of 800 nM or less (e.g. 10-800 nM, 30-600 nM or 37-558 nM, or 600 nM or less, or 558 nM or less), depending on the halogen (iodine, bromine or chloride). In a very specific embodiment of the invention, the affinity is measured by using an inhibition method (e.g. by using a Biacore 200T instrument), for example, as described in the article by Tullila and Nevanen (2017, International Journal of Molecular Sciences 18 (6), 1169).In very specific embodiments, the KD value of the recombinant antibody or antigen-binding fragment is as follows: T4 is less than 0.6+ / -0.04nM (e.g., 0.53+ / -0.04nM), halogenated bisphenol A is less than 560+ / -0.04nM, less than 70+ / -1.9nM or less than 40+ / -1.8nM (e.g., tetraiodobisphenol A (TIBPA) is less than 40+ / -1.8nM (e.g., 37.3+ / -21nM), tetrabromobisphenol A (TBBPA) is less than 70+ / -1.9nM (e.g., 69.2+ / -1.9nM) and / or tetrachlorobisphenol A (TCBPA) is less than 560+ / -21nM (e.g., 558+ / -21nM)). In one embodiment of the invention, KD is measured by competition ELISA and / or SPR sensor, for example, as described in the article by Tullila and Nevanen (2017, International Journal of Molecular Sciences 18(6), 1169), or by using a Biacore 200T device, for example, as described in the article by Tullila and Nevanen (2017, International Journal of Molecular Sciences 18(6), 1169).

[0113] In one embodiment, the recombinant antibody or antigen binding fragment of the present invention has cross-reactivity to T3 and / or T2. In a very specific embodiment, the KD value of the recombinant antibody or antigen binding fragment is as follows: T3 30+ / -0.4nM or less, 25+ / -0.4nM or less, or 21+ / -0.4nM or less (e.g., 20.8+ / -0.4nM) and / or T2 1850+ / -21nM or less (e.g., 1810+ / -21nM). In a very specific embodiment of the present invention, cross-reactivity is measured by utilizing an inhibition method (e.g., Biacore 200T device), for example, as described in Tullila and Nevanen's article (2017, International Journal of Molecular Sciences 18(6), 1169). As used herein, "cross-reactivity" refers to the reaction between an antibody or antigen binding fragment and an antigen other than the antigen (T4) that is most specific to the antibody or antigen binding fragment. In fact, cross-reactivity measures the extent to which different antigens behave similarly to antibodies or antigen-binding fragments.

[0114] In some embodiments of the invention, the antibody or antigen binding region (eg, single chain Fv or Fab fragment) is capable of binding to T4 thyroid hormone with a relative IC 50Equal to or less than 0.70 nM (e.g., 0.60 nM or less, or 0.54 nM or less (e.g., 0.54 nM as determined by ELISA)). In other embodiments, the antibody or antigen binding region is capable of binding to halogenated bisphenol A at a relative IC 50 Equal to or less than 560 nM (e.g., 550 nM or less, 75 nM or less, 55 nM or less (e.g., TIBPA 54 nM, TBBPA 74 nM, TCBPA 544 nM as determined by ELISA)). For example, the IC values ​​of T4 and tetrahalobisphenol A are 50 The values ​​may be 0.50-0.60 nM (e.g., 0.53 nM) and 37-558 nM, respectively, depending on the halogen. In a very specific embodiment of the invention, IC 50 The IC value is measured by using the inhibition method (Biacore T200 instrument), for example, as described in the article by Tullila and Nevanen (2017, International Journal of Molecular Sciences 18(6), 1169). In a competitive binding assay, IC 50 is the concentration of competing ligand that displaces 50% of specific binding to the immobilized T4-AP conjugate. In competitive binding assays, a single concentration of antibody is used in each assay tube. The ligand is used at low concentrations, typically at its K d The specific binding level of the antibody is then determined in the presence of a range of concentrations of the competing compound to measure the potency of their binding to their competing antibody. Competition curves can also be fitted to a logistic function using a computer. The IC can be converted to 50 The value was converted to absolute inhibition constant K i .

[0115] The advantages of the present invention are at least high affinity and unique specificity. In one embodiment, "high affinity" refers to an affinity for T4 thyroid hormone of 800pM or less (more specifically, for example, 600pM or less, or 530pM or less) (for example, when measured by using an inhibition method (Biacore T200 device), for example, as described in the article by Tullila and Nevanen (2017) described earlier in this disclosure. As used herein, "specificity" refers to the degree to which the recombinant antibodies or antigen-binding fragments of the present invention are able to detect the target molecule of interest (i.e., T4) relative to other thyroid hormones and halogenated bisphenol A (e.g., tetrahalobisphenol A). As used herein, "unique specificity" refers to a specific specificity value of the present invention, for example, when measured by using an inhibition method (Biacore T200) as measured, for example, as described in Tullila and Nevanen (2017). In very specific embodiments of the invention, the recombinant antibody or antigen-binding fragment has an affinity for T4 thyroid hormone of 800 pM or less (e.g., 10-800 pM or 30-600 pM or 200-530 pM, or 600 pM or less, or 530 pM or less); and has cross-reactivity to T3 or T2, with a KD value of T3 30+ / -0.4 nM or less, and / or T2 1850+ / -21 nM or less; or is capable of relative IC 50 Equal to or less than 0.70nM binding to T4 thyroid hormone. In a more specific embodiment of the present invention, the recombinant antibody or antigen-binding fragment has an affinity for T4 thyroid hormone of 800pM or less (e.g., 10-800pM or 30-600pM or 200-530pM, or 600pM or less, or 530pM or less); and has cross-reactivity to T3 or T2, with a KD value of T3 30+ / -0.4nM or less, and / or T2 1850+ / -21nM or less; and can be used with a relative IC 50 Equal to or less than 0.70nM bound to T4 thyroid hormone.

[0116] Mammalian cell lines are widely used expression systems for producing recombinant antibodies. Bacteria, yeast, filamentous fungi, plant cells and insect cells can also be used to produce recombinant antibodies, so that when the glycosylation pattern does not play a key role (for example, in in vitro diagnosis), the production cost is reduced. Therefore, the host cell comprising the recombinant nucleic acid molecule encoding the antibody or antigen-binding fragment or the expression vector comprising the recombinant nucleic acid molecule is not limited to but can be selected from the group consisting of, for example, eukaryotic or prokaryotic cells, bacteria, yeast, filamentous fungi and animals, humans, mammals, plants and insect cell lines. It can even be a hybridoma cell, which produces a recombinant monoclonal antibody after conversion.

[0117] Recombinant antibodies or Fabs can be developed, for example, by using genetically engineered genes expressed in in vitro cell lines or by techniques based on traditional hybridomas. Recombinant antibodies or Fabs can also be produced by synthesis. All of these techniques for producing recombinant antibodies are well known to those skilled in the art and are described in practical manuals and guidelines for describing laboratory molecular techniques. The recombinant techniques in producing antibodies or Fabs can produce identical antibodies in each manufacturing batch. In addition, the manufacturing process provides high purity and ultra-low batch variability.

[0118] In one embodiment of the present invention, a method for producing a recombinant antibody or antigen-binding fragment for binding to T4 thyroid hormone comprises: introducing an expression vector comprising a nucleic acid molecule encoding the antibody or antigen-binding fragment of the present invention into a host cell, and growing the cell under conditions that allow the production of the antibody or antigen-binding fragment, and optionally further recovering the produced antibody or antigen-binding fragment.

[0119] Recombinant antibody or Fab library is usually an expression library, which is typically a phage display library. The general principle of displaying recombinant antibody or Fab library is that they present the Fab as a fusion protein on a surface, which can be a surface of a microbial cell (such as a yeast or bacterial cell) or a phage. Displaying a recombinant library can also be a display library, in which a stable complex of a nascent protein and mRNA is produced in an in vitro expression system. Phage display is the most commonly used display method for antibody libraries. Easily search for antibodies or Fabs from a recombinant phage display antibody library.

[0120] Phage display antibody libraries can be constructed by encoding DNA for antibody fragments into appropriate phage display vectors.

[0121] The DNA encoding the variants of millions of antibody fragments is cloned in batches into a vector as a part of a phage coat protein. Large libraries containing millions of antibody fragments with different specificities can be obtained by transforming the vector into bacteria. The cultivation of bacteria results in the expression of phages displaying antibody fragments on the phage surface. The genes of the displayed antibodies are carried in the phagemid plasmid encapsulated in the phage, thereby connecting the genotype with the phenotype. The physical connection between the displayed protein and its DNA allows a large number of protein variants to be screened by a simple in vitro selection procedure that is referred to as panning (panning / panning), each of which is connected to its corresponding DNA.

[0122] In its simplest form, panning can be performed by incubating a pool of phage displaying a variant with an antigen or ligand of interest that has been immobilized on a support, washing away unbound phage, and eluting specifically bound phage by disrupting binding to the ligand. The eluted phage is then amplified in vivo.

[0123] This process is repeated several times, resulting in a progressively enriched phage pool that favors the tightest binding sequences. After approximately 3 to 6 rounds of selection, individual antibody clones are next subjected to screening assays, and the best clones are sequenced and examined for diversity of binders.

[0124] The present invention relates to novel T4 thyroid hormone specific recombinant antibodies. When compared with conventional monoclonal and polyclonal antibodies, the advantages of recombinant antibodies include reduced size, efficient immobilization and inexpensive bacterial production.

[0125] Antibody libraries can be used to select novel antibodies in vitro. 6-9 Different clones. Selection of the recombinant antibody library can be performed, for example, by an automatic magnetic bead processor. Several different selection methods can be used to select specific antibodies. Single recombinant antibody clones are screened, for example, by a high-throughput robotic station. Preliminary characterization of candidate antibodies is performed, for example, by ELISA and sequence comparison.

[0126] A subtype of the strain containing the antibody has been deposited with the VTT Culture Collection, PO Box 1000, FI-02044 VTT, Finland (http: / / culturecollection.vtt.fi) under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, 1977, with the following codes (E-numbers): T4 Fab / pKKtac / RV308; VTT E-173550.

[0127] The present invention also relates to rapid and simple assays for thyroid hormone (T4) (specific) and / or halogenated bisphenol A (specific) antibodies and immunoassays in various diagnostic platforms and sensors. One aspect of the present invention relates to immunoassays comprising the recombinant antibodies or antigen-binding fragments of the present invention. In addition, the present invention relates to the use of the immunoassay in the method of the present invention, i.e., a method for determining the concentration of T4 thyroid hormone and / or halogenated bisphenol A in a sample. In one embodiment of the present invention, the immunoassay further comprises additional reagents necessary for performing the immunoassay and / or instructions for performing the immunoassay. In one embodiment, the immunoassay comprises reagents necessary for performing the immunoassay, such as one or more selected from the group consisting of reaction solutions, buffers, washing solutions and detection means such as labels (e.g., enzyme labels).

[0128] Optionally, the immunoassay of the present invention may also include a control sample (e.g., a positive or negative control sample) or a reference level that reveals the specific T4 thyroid hormone and / or halogenated bisphenol A level or concentration. In addition, quality control may be optionally included in the immunoassay. In a specific embodiment, the immunoassay includes instructions for performing an immunoassay to determine the T4 thyroid hormone and / or halogenated bisphenol A concentration of a sample.

[0129] In one embodiment, the immunoassay is a competitive or non-competitive immunoassay. In other embodiments, the method for determining the concentration of T4 thyroid hormone and / or halogenated bisphenol A comprises or is a competitive or non-competitive immunoassay. Competitive immunoassays include homogeneous (e.g., fluorescence polarization assays) and heterogeneous (e.g., competitive ELISA) immunoassays. In a competitive immunoassay, an unlabeled analyte in a sample competes with a labeled analyte for binding to an antibody or antigen-binding fragment. The amount of labeled unbound analyte is then measured. The more analyte present in the sample, the less labeled analyte is detected. As used herein, "non-competitive immunoassay" refers to, for example, an immune complex assay (Pulli, Takkinen, 2005, Anal. Chem. 77, 2637-2642). In non-competitive immunoassays, the intensity of the signal is proportional to the amount of analyte in the sample.

[0130] The one or more labels optionally used in the present invention can be any conventional label, such as a radioactive label, an enzyme, a nucleotide sequence or a fluorescent compound. The immunoassay is not limited to but can be selected from a group consisting of, for example, ELISA, immuno-PCR or FIA. In one embodiment, the immunoassay for T4 thyroid hormone and / or halogenated bisphenol A can be, for example, a conventional sandwich test or lateral flow test in a microtiter well. In other embodiments, the antibodies, antigen-binding fragments and / or any engineered versions thereof included in the present invention are used in sensors based on immunoassays. In addition, any other assay type, such as agglutination tests, lateral flow tests, capillary electrophoresis, antibody arrays, cantilevers and / or microfluidic assay systems or any combination thereof, can be applied to the present invention. In fact, the method for determining the concentration of T4 thyroid hormone and / or halogenated bisphenol A may include the use of one or more of the (immuno) assays.

[0131] The detection mode of the method or immunoassay of the present invention can be any conventional detection mode, including but not limited to colorimetric, fluorescent, paramagnetic, electrochemical or label-free (eg, surface plasmon resonance and quartz crystal microbalance) detection modes.

[0132] In the method of the present invention for determining the concentration of T4 thyroid hormone and / or halogenated bisphenol A in a sample, the recombinant antibody or antigen-binding fragment is contacted with the sample, for example in the above-mentioned immunoassay. The level or amount of T4 thyroid hormone and / or halogenated bisphenol A in the sample can be determined, for example, based on colorimetric, fluorescent, paramagnetic, electrochemical or label-free (e.g., surface plasmon resonance and quartz crystal microbalance) detection modes. Optionally, the determination may also include the use of any suitable statistical method known to those skilled in the art.

[0133] The present invention also relates to a test kit comprising the recombinant antibody or antigen-binding fragment of the present invention. And, the present invention relates to the use of the test kit in the method of the present invention. In one embodiment of the present invention, the test kit may further include one or more reagents for performing the test, and optionally instructions for using or performing the test. In one embodiment, the test kit includes one or more reagents selected from the group consisting of a reaction solution, a buffer, a washing solution and a detection means such as a label and a fluorometer. In a specific embodiment of the present invention, the test kit includes at least an enzyme label. In one embodiment, the test kit is an immunoassay kit, and in a further embodiment, the immunoassay is a competitive or non-competitive immunoassay. In a very specific embodiment, the test kit includes reagents for performing the immunoassay and / or instructions for performing the immunoassay.

[0134] In a specific embodiment, the antibody or antigen-binding fragment included in the immunoassay or test kit includes the complementarity determining regions (CDRs) of the antibody light chain and heavy chain, wherein the light chain CDR region includes or has the amino acid sequence of amino acids 24-33, 49-55 and 88-96 of SEQ ID NO:3 or SEQ ID NO:5, and the heavy chain CDR region has the amino acid sequence of amino acids 31-35, 50-66 and 99-113 of SEQ ID NO:4 or SEQ ID NO:6.

[0135] Optionally, control samples (e.g., positive or negative control samples) or reference levels that reveal specific T4 thyroid hormone and / or halogenated bisphenol A levels can also be used in the present invention. Also, quality controls can be optionally included in the test kit. In a specific embodiment, the test kit includes instructions for performing a method for determining the T4 thyroid hormone and / or halogenated bisphenol concentration of a sample.

[0136] The determination or test may be performed by pre-drying the reagents (at least the antibody or antigen binding fragment) in the wells of a microtiter plate and adding a dilution series of at least one sample to the wells. In such cases, the test kit may include multiple reagent pairs physically separated from each other, such as in the form of a microarray, so that multiple antigens may be tested simultaneously from a sample. The determination or test kit of the present invention may also be applied to high-throughput determinations of a large number of samples.

[0137] In a specific embodiment, the present invention can be applied to, for example, rapid diagnosis of T4 thyroid hormone and / or halogenated bisphenol A in clinical human or animal samples. As used herein, "rapid diagnosis" refers to at least immunoassays (such as ELISA), lateral flow assays, sensor solutions, and similar applications known and permitted to the skilled person.

[0138] In one embodiment, T4 thyroid hormone and / or halogenated bisphenol A levels are determined in vitro from a sample such as a sample obtained from a subject or any biological sample. In one embodiment of the invention, the sample is a biological sample, an environmental sample, a material sample or a food sample, or a sample of a subject. In one embodiment of the invention, the subject is a human or an animal, a child, a teenager or an adult. Also, any animal, such as a pet, livestock or production animal, can be a subject of the present invention. Samples obtained from a subject include, but are not limited to, samples of blood, serum, plasma, urine, tears, sweat, secretions, biopsy or tissue.

[0139] In one embodiment, the recombinant antibodies, antigen binding fragments or immunoassays of the present invention can be used to screen for the potential of endocrine disruptors associated with the thyroid axis present in a sample. As used herein, "thyroid axis" refers to a part of the neuroendocrine system, and as the name implies, it depends on the thyroid gland. The thyroid axis regulates development, energy metabolism and growth, and the axis is controlled by complex central and peripheral signals mediated primarily by hypothalamic thyrotropin-releasing hormone (TRH) and pituitary thyroid stimulating hormone (TSH).

[0140] Before classifying a human or animal as suitable for determining T4 thyroid hormone and / or halogenated bisphenol A levels according to the present invention, the clinician may or may not perform other medical evaluations or analyses. For example, any other biomarker or level thereof may be determined before, simultaneously with, or after the methods or assays of the present invention. After these preliminary studies, and based on, for example, deviations from normal results, the clinician may recommend the methods or assays of the present invention to the subject. Alternatively, the methods and assays of the present invention are also suitable for screening small population samples as well as large population samples.

[0141] As used herein, the terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acids of any length.

[0142] As used herein, "polynucleotide" refers to any polynucleotide including a nucleic acid sequence encoding an amino acid polymer or polypeptide in question or a conservative sequence variant thereof, such as single-stranded or double-stranded DNA (genomic DNA or cDNA) or RNA. Conservative nucleotide sequence variants (i.e., nucleotide sequence modifications that do not significantly change the biological properties of the encoded polypeptide) include variants caused by genetic code degeneration and by silent mutations.

[0143] Minor changes or modifications to any one of the sequences or subsequences listed in the specification and claims are still within the scope of the invention as long as they do not affect the binding activity and / or affinity of the antibody or antigen-binding fragment. Methods for making any genetic modification are generally well known and described in various practical manuals describing laboratory molecular techniques.

[0144] Generally, the presence, absence or amount of a T4 polypeptide (or its activity) or polynucleotide in a sample can be detected by any suitable method known in the art. Non-limiting examples of suitable detection methods include enzymatic assays, PCR-based assays (e.g., qPCR, RT-PCR), immunological detection methods, and combinations thereof.

[0145] The following examples are given to further illustrate embodiments of the present invention, but are not intended to limit the scope of the present invention. For those skilled in the art, it is obvious that with the advancement of technology, the concept of the present invention can be implemented in various ways. The present invention and its embodiments are not limited to the embodiments described below, but can be changed within the scope of the claims.

[0146] Example

[0147] Example 1. Construction of antibody gene library, selection and screening of anti-T4 antibodies

[0148] Freund's adjuvant with T4- and T3- The conjugate was used to immunize mice multiple times. As described in Tullila and Nevanen (2017, International Journal of Molecular Sciences, 18 (6), 1169), the library was constructed and propagated onto the surface of bacterial phage. T4-AP was biotinylated with sulfo-NHS-SS-biotin reagent according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The biotinylated T4-AP was incubated with a Fab fragment phage display library and captured with streptavidin-coupled high-speed beads (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The magnetic particles were washed with PBST and bound to phage eluted with DTT. A total of four rounds of selection were performed before single clone screening began.

[0149] A total of 384 individual colonies were picked from the LB-amp plates and placed on 96-well microtiter plates containing 100 μl of SB-culture medium supplemented with 100 μg / ml ampicillin, 10 μg / ml tetracycline and with +1% (w / v) glucose. On the next day, 9 μl of the overnight culture was added to 100 μl of SB (IPTG, Sigma-Aldrich, St. Louis, MO, USA) / well) containing 100 μg / ml ampicillin, 10 μg / ml tetracycline, 0.1% glucose and 1 mM isopropyl-β-D-thiogalactopyranoside and grown overnight at 34°C, 80% humidity with shaking. For the ELISA screening assay, 300 ng of T4-HSA in pH 9.6 sodium bicarbonate buffer was coated onto each well of a MaxiSorp plate (Nunc) overnight at +4°C. On the next day, the plates were washed with PBST and (Thermo Fisher Scientific) for 30 minutes and then washed again. Dilute 1:1 in 5% paraformaldehyde and transfer 100 μl to each well. The samples were incubated for 1 hour and the wells were then washed as before. Anti-mouse κ-AP (Southern Biotech) diluted 1:2000 in 5% paraformaldehyde was added to the wells, incubated for 1 hour and washed as before. 2 The detection was performed with 2 mg / ml p-nitrophenyl phosphate (pNPP, Sigma-Aldrich) in 1% para-nitrophenyl phosphate (Reagena, Toivala, Finland). After 30 minutes of enzyme reaction, the wells were read at A405 using a plate reader (Beckmann Coulter DTX 880 multimode detector). The clone (T4) that gave the highest intensity on T4- and T3-HSA-coated wells was selected for further characterization.

[0150] The antibody encoding insert was transferred into the pKKtac expression vector (Takkinen, K. et al., Protein Engineering, Design and Selection, 4(7), 837-841), which includes a His at the C-terminal position of the Fab heavy chain. 6 The construct was further transferred into RV308 Escherichia coli strain (ATCC 31608), and T4 Fab fragment antibodies were produced and purified as described in Tullila and Nevanen (2017, International Journal of Molecular Sciences, 18(6), 1169) (see Figures 1A (SEQ ID NO: 1), 1B (SEQ ID NO: 2), 2A (SEQ ID NO: 3) and 2B (SEQ ID NO: 4)).

[0151] Example 2. Affinity and specificity determination

[0152] The affinity of the purified anti-T4 Fab fragments for various analytes was analyzed using a surface plasmon resonance instrument BIAcore T200 (GE Healthcare, Piscataway, NJ, USA) and inhibition assays (Nieba, L. et al. 1996, Anal. Biochem. 234, 155-165; Badescu, GO et al. 2016, PLoS ONE, 11, e0152148; Persson, BD et al. 2009, J. Virol. 83, 673-686; Tullila, A., and Nevanen, TK 2017, International Journal of Molecular Sciences, 18(6), 1169; Adamczyk, M. et al. 1998, Bioconjugate chemistry, 9(1), 23-32. Prior to immobilization, the BIAcore was ionized as described by Cannon et al. (2004, Analytical biochemistry, 330.1:98-113). The surface of a CM5 sensor chip (29104988, GE Healthcare, Piscataway, NJ, USA) was pretreated. T4-AP and AP (P5931, Sigma-Aldrich, St. Louis, MO, USA) were diluted to a concentration of 100 μg / mL in 10 mM sodium acetate pH 4.0 (BR100351, GE Healthcare, Piscataway, NJ, USA). T4-AP and AP were immobilized to the pretreated CM5 chip using EDC / NHS-chemistry according to the manufacturer's instructions. The concentration of the anti-T4 Fab fragment was calculated using the theoretical extinction coefficient of the anti-T4 Fab fragment (ExPASy ProtParam tool, http: / / web.expasy.org / protparam) and by measuring A280 with a NanoDrop 2000c instrument (Thermo Disher Scientific, Waltham, MA, USA). 4 nM anti-T4 Fab fragments were prepared into 2% (v / v) DMSO-PBSP (20 mM phosphatase, 2.7 mM KCl, 137 mM NaCl, 0.05% Tween 20 (prepared from 10×PBSP+, 28-9950-84, GE Healthcare, Piscataway, NJ, USA) supplemented with DMSO (D5897, Sigma-Aldrich, St. Louis, MO, USA) and further diluted 3 / 5-fold into the same buffer to obtain a calibration series of free Fab fragment antibody. A total of eight dilutions were used in each analysis. The contact time for association was set to 420 seconds and for dissociation to 30 seconds.Successful regeneration of anti-T4 Fab was obtained with 10 mM NaOH supplemented with 0.05% (v / v) P20. The flow rate was set at 30 μl / min during all cycling steps.

[0153] Different concentrations of analytes T4 (T2376, Sigma-Aldrich, St. Louis, MO, USA), T3 (T2877, Sigma-Aldrich, St. Louis, MO, USA), T2 (D0629, Sigma-Aldrich, St. Louis, MO, USA), T0 (T5905, Sigma-Aldrich, St. Louis, MO, USA), tetraiodobisphenol A (4971-59-9, Spectra Group Limited), tetrabromobisphenol A (330396, Sigma-Aldrich, St. Louis, MO, USA), tetrachlorobisphenol A (TCI-T0062, Tokio Chemical Industry,) and bisphenol A (239658, Sigma-Aldrich, St. Louis, MO, USA) were pre-incubated with 2 nM concentration of anti-T4 Fab fragment. The pre-incubated mixture was injected onto the sensor chip surface for seven minutes and dissociated for 30 seconds. The bound anti-T4 Fab fragment was successfully regenerated from the surface using 10 mM NaOH + 0.05% (v / v) Tween 20. The data collection point was set to 10 seconds after the start of the dissociation phase. All sample series were performed in triplicate, with a single sample series consisting of increasing concentrations of the small molecule in the pre-incubation mixture. The data were subtracted from the reference and blank. In addition, small differences in DMSO between samples were compensated using a DMSO solvent correction series as recommended by the instrument manufacturer (GE Healthcare). The concentration of free anti-T4 Fab fragment remaining in the pre-incubated mixture was calculated by comparing the obtained responses relative to a standard curve of anti-T4 Fab fragments. Affinity constants were calculated using a four-parameter logistic function using the BIAcore T200 evaluation software (GE Healthcare, Piscataway, NJ, USA). (See Figure 4A and B and Table 1. )

[0154] Table 1. Affinity constants using BIAcore T200 evaluation software

[0155]

[0156] Example 3. ELISA assay

[0157] L-Thyroxine

[0158] Microtiter plate wells (Nunc, 436023) were coated with 100 μl of 4 μg / ml T4-BSA (Fitzgerald, 80-IT50) in PBS (15 mM phosphate, 150 mM NaCl, pH 7.3) for one hour at +37°C with shaking at 600 rpm. The wells were then washed three times with 300 μl PBS supplemented with 0.05% (v / v) Tween 20 (Sigma-Aldrich, 93773-250G). The wells were then washed with 200 μl of PBS at room temperature with shaking at 600 rpm. (Thermo Fisher Scientific, 37516) blocked for 30 minutes and washed as above. 100 μl of the pre-incubated mixture, containing 0.25 nM anti-T4 Fab and different concentrations of L-thyroxine (T4) (Sigma-Aldrich, T2376) in PBSP (GE Healthcare, 28-9950-84) supplemented with 1% DMSO (v / v) (Sigma-Aldrich, D5879-100ML), was transferred to the wells and incubated at room temperature for 30 minutes at 600 rpm. Each sample has 12 replicates. The wells were washed as above, and 100 μl of goat anti-mouse κ-HRP (SoutherBiotech, 1050-05) diluted in Superblock at 1:1000 was transferred to the wells and incubated at room temperature for 30 minutes at 600 rpm. The wells were washed as above, and 100 μl of TMB Microwell Peroxidase Substrate System (2-C) (SeraCare, 50-76-00) was transferred to the wells and the enzymatic reaction was carried out at room temperature with shaking at 600 rpm for 30 minutes. 3 PO 4 The reaction was stopped by transfer into the wells and measured at A450 using Varioskan (Thermo Fisher Scientific). Data were analyzed using OriginPro 2017–software.

[0159] like Figure 5 As seen in Figure 2, in the competitive ELISA assay, the limit of detection (LOD) value was 84 pM and the limit of quantification (LOQ) was 314 pM, indicating the potential of T4 Fab for measuring total thyroxine concentration in samples (see Figure 5 results).

[0160] Tetraiodobisphenol A

[0161] The assay was performed as in the preceding paragraph (under the heading L-thyroxine), except that TIBPA (tetraiodobisphenol A) was used instead of T4 (thyroxine).

[0162] The competitive ELISA results for LOD and LOQ were comparable to the Biacore affinity measurements. The sensitivity of the competitive ELISA assay for TIBPA was two orders of magnitude lower than that for L-thyroxine. Figure 6 The results in .

[0163] Tetrabromobisphenol A

[0164] The assay was performed as in the paragraph under the heading L-thyroxine, except that TBBPA (tetrabromobisphenol A) was used instead of T4 (thyroxine).

[0165] The competitive ELISA results for LOD and LOQ were comparable to the Biacore affinity measurements. The sensitivity of the competitive ELISA assay for TBBPA was more than two orders of magnitude lower than that for L-thyroxine. Figure 7 The results in .

[0166] Tetrachlorobisphenol A

[0167] The assay was performed as in the paragraph under the heading L-thyroxine, except that TCBPA (tetrachlorobisphenol A) was used instead of T4 (thyroxine).

[0168] The competitive ELISA results for LOD and LOQ were comparable to the Biacore affinity measurements. The sensitivity of the competitive ELISA assay for TCBPA was three orders of magnitude lower than that for L-thyroxine. Figure 8 The results in .

[0169] Bisphenol A

[0170] The assay was performed as in the paragraph under the heading L-thyroxine, except that BPA (bisphenol A) was used instead of T4 (thyroxine).

[0171] In the competitive ELISA assay, no competition was observed even at high concentrations (5 μM) of BPA. Fig. 9 The results in .

[0172] Example 4. Three-dimensional structure of anti-T4 antibody

[0173] Crystallization of T4-Fab and T4 was performed by vapor diffusion in a hanging drop consisting of 2 μl of T4-Fab (4 mg / ml, previously incubated with T4) and 2 μl of a reservoir solution. Initial crystals of the T4-Fab / T4 complex were obtained at 20°C in a reservoir solution of 0.1 M ammonium sulfate, 0.1 M HEPES, pH 7.2 and 25% (w / v) PEG3350. Streak seeding was used to improve the crystal quality. The crystals were cryoprotected by immersion in a crystallization solution supplemented with 20% glycerol and then rapid freezing in liquid nitrogen. Data for the structure of the T4-Fab / T4 complex were collected at 100 K on beamline I04 at the Diamond Synchrotron and on beamline ID23 at the ESRF (Grenoble, France), respectively. The data sets were processed with XDS (Kabsch) and scaled with XSCALE. The crystals belong to space group C2 and have one molecule per asymmetric unit. The high-resolution structure was solved by molecular replacement with PHASER (McCoy, AJ et al. J. Appl. Cryst. 40, 658-674 (2007)) by using the coordinates of the Fab hGR-2F6 (PDB-code 1DQD) as a search model. Several cycles of construction and refinement were performed with COOT (Emsley, P., Lohkamp, ​​B., Scott, WG & Cowtan, K. Acta Cryst. D 66, 486-501 (2010)) and PHENIX (Adams, PD et al. Acta Cryst. D, 66, 213-221 (2010)). The three-dimensional structure was examined with a suitable molecular graphics software. The overall structure of T4-Fab in complex with T4 is shown in Figure 11a, and a detailed picture on the active site is shown in Figure 11b. Figure 10a reveals that the atoms of the ligand and the anti-T4 Fab are up to The atomic distances of 1 and 2 indicate the molecular basis of the binding of T4 and TIBPA ligands. Detailed pictures of halogen bonding in the structures of anti-T4 Fab and T4 or tetraiodobisphenol A (TIBPA) are shown in Figure 10b 1 and 2, respectively. Sequence Listing <110> Technical Research Centre of Finland (VTT Oy) <120> Anti-thyroid hormone (T4) recombinant antibody or antigen-binding fragment <130> PPI20031421FI <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 639 <212> DNA <213> Artificial sequence <220> <223> T4 Fab light chain nucleotide sequence <400> 1 gatattgttc tcaaccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60 atgacctgca attccagctc aagtgtaagt tacatgcact ggtaccagca taagccagga 120 tcctcccccc gactcctgat ttatgacaca tccaacctgg cttctggagt ccctgctcgc 180 ttcagtggca gtggatctgg gacctcttac tctctcacaa tcagccgaat ggaggctgaa 240 gatgctgcca cttattactg ccagcaaagg agtagttacc cgctcacgtt cggtgctggg 300 accaagctgg aaataacccg ggctgatgct gcaccaactg tatccatctt cccaccatcc 360 agtgagcagt taacatctgg aggtgcctca gtcgtgtgct tcttgaacaa cttctacccc 420 aaagacatca atgtcaagtg gaagattgat ggcagtgaac gacaaaatgg cgtcctgaac 480 agttggactg atcaggacag caaagacagc acctacagca tgagcagcac cctcacgctc 540 accaaggacg agtatgaacg acataacagc tatacctgtg aggccactca caagacatca 600 acttcaccca ttgtcaagag cttcaacagg aatgagtgt 639 <210> 2 <211> 678 <212> DNA <213> Artificial Sequence <220> <223> T4 Fab heavy chain nucleotide sequence <400> 2 gaagtgaagc ttgaggagtc tgggggaggc ttagtgaaac ttggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt agctattaca tgtcttgggt tcgccagact 120 ccagagaaga ggctggagtt ggtcgcagcc attaatagta atggtggtag cacctactat 180 ccagacactg tgaagggccg attcaccatc tccagagaca atgccaagaa caccctgtac 240 ctgcaaatga gcagtctgaa gtctgaggac acagccttgt attactgtgc aagaggggat 300 tactacggta gtagcttata ttactatgct atggactact ggggtcaagg aacctcagtc 360 accgtctcct cagccaaaac gacaccccca tctgtctatc cactggcccc tggatctgct 420 gcccaaacta actccatggt gaccctggga tgcctggtca agggctattt ccctgagcca 480 gtgacagtga cctggaactc tggatccctg tccagcggtg tgcacacctt cccagctgtc 540 ctgcagtctg acctctacac tctgagcagc tcagtgactg tcccctccag cacctggccc 600 agcgagaccg tcacctgcaa cgttgcccac ccggccagca gcaccaaggt ggacaagaaa 660 attgtgccca gggattgt 678 <210> 3 <211> 213 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of T4 Fab light chain <400> 3 Asp Ile Val Leu Asn Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Asn Ser Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln His Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Ile Thr Arg Ala Asp Ala Ala Pro 100 105 110 Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly 115 120 125 Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn 130 135 140 Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn 145 150 155 160 Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser 165 170 175 Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr 180 185 190 Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe 195 200 205 Asn Arg Asn Glu Cys 210 <210> 4 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> Amino Acid Sequence of T4 Fab Heavy Chain <400> 4 Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Leu Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Leu Val 35 40 45 Ala Ala Ile Asn Ser Asn Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Tyr Tyr Gly Ser Ser Leu Tyr Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr 115 120 125 Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn 130 135 140 Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val 180 185 190 Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val 195 200 205 Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg 210 215 220 Asp Cys 225 <210> 5 <211> 213 <212> PRT <213> Artificial sequence <220> <223> T4 Fab light chain amino acid sequence <220> <221> misc_feature <222> (93)..(93) <223> Xaa is Tyr or Phe <400> 5 Asp Ile Val Leu Asn Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Asn Ser Ser Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln His Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Xaa Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Ile Thr Arg Ala Asp Ala Ala Pro 100 105 110 Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly 115 120 125 Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn 130 135 140 Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn 145 150 155 160 Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser 165 170 175 Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr 180 185 190 Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe 195 200 205 Asn Arg Asn Glu Cys 210 <210> 6 <211> 226 <212> PRT <213> Artificial sequence <220> <223> T4 Fab heavy chain amino acid sequence <220> <221> misc_feature <222> (33)..(33) <223> Xaa is Tyr or Phe <220> <221> misc_feature <222> (59)..(59) <223> Xaa is Tyr or Phe <220> <221> misc_feature <222> (107)..(107) <223> Xaa is Tyr or Phe <220> <221> misc_feature <222> (109)..(109) <223> Xaa is Tyr or Phe <220> <221> misc_feature <222> (113)..(113) <223> Xaa is Tyr or Phe <400> 6 Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Leu Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Xaa Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Leu Val 35 40 45 Ala Ala Ile Asn Ser Asn Gly Gly Ser Thr Xaa Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Tyr Tyr Gly Ser Ser Leu Xaa Tyr Xaa Ala Met Asp 100 105 110 Xaa Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr 115 120 125 Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn 130 135 140 Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val 180 185 190 Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val 195 200 205 Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg 210 215 220 Asp Cys 225 <210> 7 <211> 639 <212> DNA <213> Artificial sequence <220> <223> T4 Fab light chain nucleotide sequence <220> <221> misc_feature <222> (277)..(279) <223> n is a, c, g or t <400> 7 gatattgttc tcaaccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60 atgacctgca attccagctc aagtgtaagt tacatgcact ggtaccagca taagccagga 120 tcctcccccc gactcctgat ttatgacaca tccaacctgg cttctggagt ccctgctcgc 180 ttcagtggca gtggatctgg gacctcttac tctctcacaa tcagccgaat ggaggctgaa 240 gatgctgcca cttattactg ccagcaaagg agtagtnnnc cgctcacgtt cggtgctggg 300 accaagctgg aaataacccg ggctgatgct gcaccaactg tatccatctt cccaccatcc 360 agtgagcagt taacatctgg aggtgcctca gtcgtgtgct tcttgaacaa cttctacccc 420 aaagacatca atgtcaagtg gaagattgat ggcagtgaac gacaaaatgg cgtcctgaac 480 agttggactg atcaggacag caaagacagc acctacagca tgagcagcac cctcacgctc 540 accaaggacg agtatgaacg acataacagc tatacctgtg aggccactca caagacatca 600 acttcaccca ttgtcaagag cttcaacagg aatgagtgt 639 <210> 8 <211> 705 <212> DNA <213> Artificial sequence <220> <223> T4 Fab heavy chain nucleotide sequence <220> <221> misc_feature <222> (97)..(99) <223> n is a, c, g or t <220> <221> misc_feature <222> (175)..(177) <223> n is a, c, g or t <220> <221> misc_feature <222> (319)..(321) <223> n is a, c, g or t <220> <221> misc_feature <222> (325)..(327) <223> n is a, c, g or t <400> 8 gaagtgaagc ttgaggagtc tgggggaggc ttagtgaaac ttggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt agctatnnna tgtcttgggt tcgccagact 120 ccagagaaga ggctggagtt ggtcgcagcc attaatagta atggtggtag caccnnntat 180 ccagacactg tgaagggccg attcaccatc tccagagaca atgccaagaa caccctgtac 240 ctgcaaatga gcagtctgaa gtctgaggac acagccttgt attactgtgc aagaggggat 300 tactacggta gtagcttann ntacnnngct atggactact ggggtcaagg aacctcagtc 360 accgtctcct cagccaaaac gacaccccca tctgtctatc cactggcccc tggatctgct 420 gcccaaacta actccatggt gaccctggga tgcctggtca agggctattt ccctgagcca 480 gtgacagtga cctggaactc tggatccctg tccagcggtg tgcacacctt cccagctgtc 540 ctgcagtctg acctctacac tctgagcagc tcagtgactg tcccctccag cacctggccc 600 agcgagaccg tcacctgcaa cgttgcccac ccggccagca gcaccaaggt ggacaagaaa 660 attgtgccca gggattgtgc ggccgcacat catcatcatc atcat 705

Claims

1. A recombinant antibody or antigen-binding fragment thereof for binding to T4 thyroid hormone and for binding to halogenated bisphenol A, wherein the recombinant antibody or antigen-binding fragment comprises the following CDR amino acids: The light chain variable region CDR1 is amino acids 24-33 of SEQ ID NO: 3, The light chain variable region CDR2 is amino acids 49-55 of SEQ ID NO: 3 The light chain variable region CDR3 is amino acids 88-96 of SEQ ID NO: 3, The heavy chain variable region CDR1 is amino acids 31-35 of SEQ ID NO: 4, The heavy chain variable region CDR2 is amino acids 50-66 of SEQ ID NO: 4, and The heavy chain variable region CDR3 is amino acids 99-113 of SEQ ID NO: 4, and wherein: Y93 and L95 of SEQ ID NO: 3; Y33, A50, Y59, D100, Y107, Y109 and M111 of SEQ ID NO: 4 are responsible for binding to T4.

2. The recombinant antibody or antigen-binding fragment according to claim 1, wherein The affinity of the recombinant antibody or antigen-binding fragment to T4 thyroid hormone is 530 pM or less.

3. The recombinant antibody or antigen-binding fragment according to claim 1, wherein: The affinity of the recombinant antibody or antigen-binding fragment to halogenated bisphenol A is 600 nM or less.

4. The recombinant antibody or antigen-binding fragment according to any one of claims 1 to 3, wherein The antigen binding fragment is a single chain Fv (scFv) or a Fab fragment.

5. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the recombinant antibody or antigen-binding fragment of any one of claims 1-4.

6. The isolated nucleic acid molecule of claim 5, comprising a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO: 1 or 2.

7. An expression vector comprising the nucleic acid molecule according to claim 5 or 6.

8. A host cell comprising the nucleic acid molecule or expression vector according to any one of claims 5 to 7.

9. A method for producing a recombinant antibody or an antigen-binding fragment thereof for binding T4 thyroid hormone and for binding halogenated bisphenol A according to any one of claims 1 to 4, wherein: The method comprises introducing the expression vector of claim 7 into a host cell and growing the cell under conditions that allow production of the antibody or antigen-binding fragment.

10. The method according to claim 9, wherein: The antibodies or antigen-binding fragments produced are also recovered.

11. A test kit comprising the recombinant antibody or antigen-binding fragment according to any one of claims 1 to 4.

12. The test kit of claim 11, further comprising one or more reagents for performing the test, and optionally instructions for performing the test.

13. The test kit according to claim 11 or 12, wherein: The test kit is an immunoassay kit.

14. The test kit according to claim 13, wherein: The immunoassay is a competitive or non-competitive immunoassay.

15. The test kit according to claim 13 or 14, wherein: The test kit includes reagents for performing the immunoassay and / or instructions for performing the immunoassay.

16. A method for determining the concentration of T4 thyroid hormone and / or halogenated bisphenol A in a sample for non-diagnostic purposes, wherein: The method comprises allowing the recombinant antibody or antigen-binding fragment of any one of claims 1 to 4 to contact a sample, and then determining the concentration of T4 thyroid hormone and / or halogenated bisphenol A in the sample, respectively.

17. The method according to claim 16, wherein: The method is either a competitive or non-competitive immunoassay.

18. Use of the recombinant antibody or antigen-binding fragment according to any one of claims 1 to 4 in the preparation of a kit for determining the concentration of T4 thyroid hormone and / or halogenated bisphenol A in a sample.

19. A method for processing a sample for non-diagnostic purposes, wherein: The method comprises allowing the recombinant antibody or antigen-binding fragment of any one of claims 1 to 4 to contact a sample.

20. The method according to claim 16 or 19, wherein: The sample is an environmental sample, a material sample or a food sample.

21. Use of the recombinant antibody or antigen-binding fragment according to any one of claims 1 to 4 in the preparation of a kit for processing a sample.

22. The use according to claim 18 or 21, wherein The sample is a biological sample, an environmental sample, a material sample or a food sample.

23. The use according to claim 18 or 21, wherein The sample is a sample from a subject.

24. A method of immunoassay for non-diagnostic purposes, comprising the recombinant antibody or antigen-binding fragment according to any one of claims 1 to 4.

25. The method according to claim 24, wherein: The immunoassay is a competitive or non-competitive immunoassay.

26. Use of the recombinant antibody or antigen-binding fragment according to any one of claims 1 to 4 in the preparation of a kit for immunoassay.

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