Monoclonal antibody or antigen-binding fragment thereof for detecting folic acid and its binding protein complex, preparation method and application thereof

Abstract

The present invention relates to a monoclonal antibody or an antigen-binding fragment thereof for detecting folic acid and its binding protein complex, a preparation method and application thereof, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises: (1) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively. The monoclonal antibody or the antigen-binding fragment thereof of the present invention can bind to folic acid and its protein complex with high specificity and high affinity, thereby improving the detection accuracy and sensitivity of folic acid, thereby breaking through the limitations of low sensitivity and poor clinical relevance of traditional competitive detection reagents, and realizing sandwich immunoassay of folic acid.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibody preparation and immunological detection, and in particular to a monoclonal antibody or an antigen-binding fragment thereof for detecting folic acid and its binding protein complex, and a preparation method and application thereof. Background Art

[0002] Folic acid (Folate) is a water-soluble vitamin. After being ingested by the human body, it is reduced to dihydrofolate (DHF) and tetrahydrofolate (THF) under the double reduction action of dihydrofolate reductase. 5-methyltetrahydrofolate (5MeTHF) is the main metabolite of THF, accounting for about 82%~93% of the content in the human body. Folic acid (Folate) participates in the metabolism of one-carbon units in the body. Folic acid (Folate) deficiency is prone to cause neural tube defects, megaloblastic anemia, cardiovascular diseases and tumor diseases.

[0003] At present, the main methods for detecting folic acid include microbiological assay (MA), folate-binding protein (FBP), high performance liquid chromatography (HPLC) and ID-LC-MS / MS. These methods can detect folic acid in serum / plasma and red blood cells. In clinical practice, folic acid is mainly detected by FBP method, which has low detection cost but poor specificity. There are large differences between different detection systems, which can easily lead to misjudgment of results.

[0004] In view of the above-mentioned problems in the existing technology, there is an urgent need to develop highly specific and high-affinity monoclonal antibodies against folic acid and its binding protein complexes to meet the needs of immunological detection, so as to break through the limitations of traditional competitive methods and realize the sandwich method for detecting folic acid (Folate). Summary of the invention

[0005] The purpose of the present invention is to provide a highly specific and high affinity monoclonal antibody or an antigen-binding fragment thereof for folic acid and its binding protein complex, and to develop a high-performance antibody suitable for sandwich immunological detection with the binding protein, which has a higher sensitivity for the detection of folic acid (Folate). In addition, the antibody can also detect various folic acid metabolites in the body, that is, the total folic acid content.

[0006] In order to achieve the above objectives, the present invention has obtained, through a large number of screenings, highly specific and high affinity monoclonal antibodies or antigen-binding fragments thereof against folic acid and its binding protein complexes, their related nucleic acids and cell products, their preparation methods and applications, and immunoassay reagents based thereon, thereby providing a basis for highly specific and highly sensitive immunoassays of folic acid (Folate).

[0007] Specifically, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof against folic acid (Folate) and its binding protein complex, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises:

[0009] (1) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively; or,

[0010] (2) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, respectively; or,

[0011] (3) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO.21, SEQ ID NO.22 and SEQ ID NO.23, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO.24, SEQ ID NO.25 and SEQ ID NO.26, respectively; or,

[0012] (4) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO.31, SEQ ID NO.32 and SEQ ID NO.33, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO.34, SEQ ID NO.35 and SEQ ID NO.36, respectively; or,

[0013] (5) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO.41, SEQ ID NO.42 and SEQ ID NO.43, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO.44, SEQ ID NO.45 and SEQ ID NO.46, respectively; or,

[0014] (6) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO.51, SEQ ID NO.52 and SEQ ID NO.53, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO.54, SEQ ID NO.55 and SEQ ID NO.56, respectively; or,

[0015] (7) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO.61, SEQ ID NO.62 and SEQ ID NO.63, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO.64, SEQ ID NO.65 and SEQ ID NO.66, respectively; or,

[0016] (8) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO.71, SEQ ID NO.72 and SEQ ID NO.73, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO.74, SEQ ID NO.75 and SEQ ID NO.76, respectively; or,

[0017] (9) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO.81, SEQ ID NO.82 and SEQ ID NO.83, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO.84, SEQ ID NO.85 and SEQ ID NO.86, respectively; or,

[0018] (10) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO.91, SEQ ID NO.92 and SEQ ID NO.93, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO.94, SEQ ID NO.95 and SEQ ID NO.96, respectively.

[0019] In a preferred embodiment, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0020] (1) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO.7; and a light chain variable region having the amino acid sequence shown in SEQ ID NO.8; or,

[0021] (2) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO.17; and a light chain variable region having the amino acid sequence shown in SEQ ID NO.18; or,

[0022] (3) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO.27; and a light chain variable region having the amino acid sequence shown in SEQ ID NO.28; or,

[0023] (4) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO.37; and a light chain variable region having the amino acid sequence shown in SEQ ID NO.38; or,

[0024] (5) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO.47; and a light chain variable region having the amino acid sequence shown in SEQ ID NO.48; or,

[0025] (6) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO.57; and a light chain variable region having the amino acid sequence shown in SEQ ID NO.58; or,

[0026] (7) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO.67; and a light chain variable region having the amino acid sequence shown in SEQ ID NO.68; or,

[0027] (8) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO.77; and a light chain variable region having the amino acid sequence shown in SEQ ID NO.78; or,

[0028] (9) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO.87; and a light chain variable region having the amino acid sequence shown in SEQ ID NO.88; or,

[0029] (10) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO.97; and a light chain variable region having the amino acid sequence shown in SEQ ID NO.98.

[0030] In a further preferred embodiment, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0031] (1) a heavy chain, the amino acid sequence of which is shown in SEQ ID NO.9; a light chain, the amino acid sequence of which is shown in SEQ ID NO.10, or,

[0032] (2) a heavy chain, whose amino acid sequence is shown in SEQ ID NO.19; a light chain, whose amino acid sequence is shown in SEQ ID NO.20, or,

[0033] (3) a heavy chain, whose amino acid sequence is shown in SEQ ID NO.29; a light chain, whose amino acid sequence is shown in SEQ ID NO.30, or,

[0034] (4) a heavy chain, whose amino acid sequence is shown in SEQ ID NO.39; a light chain, whose amino acid sequence is shown in SEQ ID NO.40, or,

[0035] (5) a heavy chain, whose amino acid sequence is shown in SEQ ID NO.49; a light chain, whose amino acid sequence is shown in SEQ ID NO.50, or,

[0036] (6) a heavy chain, whose amino acid sequence is shown in SEQ ID NO.59; a light chain, whose amino acid sequence is shown in SEQ ID NO.60, or,

[0037] (7) a heavy chain, whose amino acid sequence is shown in SEQ ID NO.69; a light chain, whose amino acid sequence is shown in SEQ ID NO.70, or,

[0038] (8) a heavy chain, whose amino acid sequence is shown in SEQ ID NO.79; a light chain, whose amino acid sequence is shown in SEQ ID NO.80, or,

[0039] (9) a heavy chain, whose amino acid sequence is shown in SEQ ID NO.89; a light chain, whose amino acid sequence is shown in SEQ ID NO.90, or,

[0040] (10) A heavy chain, whose amino acid sequence is shown in SEQ ID NO.99; a light chain, whose amino acid sequence is shown in SEQ ID NO.100.

[0041] In some feasible embodiments, the antigen-binding fragment of the monoclonal antibody is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single chain antibody, human antibody, chimeric antibody or bispecific or multispecific antibody.

[0042] In a second aspect, the present invention provides a polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect. The polynucleotide is not limited by the method of its production and can be obtained by genetic engineering recombination technology or chemical synthesis method.

[0043] In a third aspect, the present invention provides a nucleic acid construct comprising the polynucleotide as described in the second aspect above, and, optionally, at least one expression control element operably linked to the polynucleotide.

[0044] In a fourth aspect, the present invention provides a recombinant vector comprising the polynucleotide as described in the second aspect above, or the nucleic acid construct as described in the third aspect above.

[0045] The vector of the present invention may be a cloning vector or an expression vector, for example, it may be a plasmid, a cosmid, a phage, etc.

[0046] In some preferred embodiments, the recombinant vector is a recombinant expression vector, preferably a eukaryotic expression vector.

[0047] In a fifth aspect, the present invention provides a transformed host cell, wherein the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, or the recombinant vector as described in the fourth aspect is transformed;

[0048] The host cell includes, but is not limited to, prokaryotic cells, such as Escherichia coli cells, eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The host cell can also be a cell line, such as a 293T cell line.

[0049] Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

[0050] In a sixth aspect, the present invention provides the use of the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect, the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, the recombinant vector as described in the fourth aspect and / or the transformed host cell as described in the fifth aspect in the preparation of a detection reagent or kit for detecting folic acid.

[0051] In a feasible embodiment, the sample is a biological sample of a subject, preferably a serum or plasma sample of a subject.

[0052] In a seventh aspect, the present invention provides use of any monoclonal antibody or antigen-binding fragment thereof selected from the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above in the preparation of a detection reagent or kit for detecting folic acid.

[0053] Preferably, the monoclonal antibody or antigen-binding fragment thereof is selected from:

[0054] (I) Antibody 1 or an antigen-binding fragment thereof;

[0055] (II) Antibody 2 or an antigen-binding fragment thereof;

[0056] (III) Antibody 3 or an antigen-binding fragment thereof;

[0057] (IV‌ ) Antibody 4 or an antigen-binding fragment thereof;

[0058] (V) Antibody 5 or an antigen-binding fragment thereof;

[0059] (VI) Antibody 6 or an antigen-binding fragment thereof;

[0060] (VII‌) Antibody 7 or an antigen-binding fragment thereof;

[0061] (VIII‌) Antibody 8 or an antigen-binding fragment thereof;

[0062] (IX) Antibody 9 or an antigen-binding fragment thereof;

[0063] (X) antibody 10 or an antigen-binding fragment thereof;

[0064] Wherein, antibody 1 or its antigen-binding fragment is defined by any item (1) of the first aspect above, antibody 2 or its antigen-binding fragment is defined by any item (2) of the first aspect above, antibody 3 or its antigen-binding fragment is defined by any item (3) of the first aspect above, antibody 4 or its antigen-binding fragment is defined by any item (4) of the first aspect above, antibody 5 or its antigen-binding fragment is defined by any item (5) of the first aspect above, antibody 6 or its antigen-binding fragment is defined by any item (6) of the first aspect above, antibody 7 or its antigen-binding fragment is defined by any item (7) of the first aspect above, antibody 8 or its antigen-binding fragment is defined by any item (8) of the first aspect above, antibody 9 or its antigen-binding fragment is defined by any item (9) of the first aspect above, and antibody 10 or its antigen-binding fragment is defined by any item (10) of the first aspect above.

[0065] In an eighth aspect, the present invention provides a detection reagent or kit for detecting folic acid (Folate), which comprises the monoclonal antibody or its antigen-binding fragment as described in the first aspect above, the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, the recombinant vector as described in the fourth aspect above and / or the transformed host cell as described in the fifth aspect above.

[0066] Preferably, the detection reagent or kit comprises the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, preferably comprises any one monoclonal antibody or antigen-binding fragment thereof selected from the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, more preferably comprises a monoclonal antibody or antigen-binding fragment thereof selected from the following:

[0067] (I) Antibody 1 or an antigen-binding fragment thereof;

[0068] (II) Antibody 2 or an antigen-binding fragment thereof;

[0069] (III) Antibody 3 or an antigen-binding fragment thereof;

[0070] (IV‌) Antibody 4 or an antigen-binding fragment thereof;

[0071] (V) Antibody 5 or an antigen-binding fragment thereof;

[0072] (VI) Antibody 6 or an antigen-binding fragment thereof;

[0073] (VII‌) Antibody 7 or an antigen-binding fragment thereof;

[0074] (VIII‌) Antibody 8 or an antigen-binding fragment thereof;

[0075] (IX) Antibody 9 or an antigen-binding fragment thereof;

[0076] (X) antibody 10 or an antigen-binding fragment thereof;

[0077] Wherein, antibody 1 or its antigen-binding fragment is defined by any item (1) of the first aspect above, antibody 2 or its antigen-binding fragment is defined by any item (2) of the first aspect above, antibody 3 or its antigen-binding fragment is defined by any item (3) of the first aspect above, antibody 4 or its antigen-binding fragment is defined by any item (4) of the first aspect above, antibody 5 or its antigen-binding fragment is defined by any item (5) of the first aspect above, antibody 6 or its antigen-binding fragment is defined by any item (6) of the first aspect above, antibody 7 or its antigen-binding fragment is defined by any item (7) of the first aspect above, antibody 8 or its antigen-binding fragment is defined by any item (8) of the first aspect above, antibody 9 or its antigen-binding fragment is defined by any item (9) of the first aspect above, and antibody 10 or its antigen-binding fragment is defined by any item (10) of the first aspect above.

[0078] In a feasible embodiment, the kit comprises a time-resolved fluorescent immunochromatography reagent, which is based on the principle of sandwich method and uses any one monoclonal antibody or antigen-binding fragment thereof selected from the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above and a folate binding protein as a capture and a detection, respectively;

[0079] Preferably, the time-resolved fluorescent immunochromatography reagent uses (1) any one of antibody 1 or its antigen-binding fragment and folate binding protein as a capture, and the other as a detection; further preferably, the time-resolved fluorescent immunochromatography reagent uses antibody 1 or its antigen-binding fragment as a capture, and folate binding protein as a detection;

[0080] Preferably, the time-resolved fluorescent immunochromatography reagent uses (2) any one of antibody 2 or its antigen-binding fragment and folate binding protein as a capture, and the other as a detection; further preferably, the time-resolved fluorescent immunochromatography reagent uses antibody 2 or its antigen-binding fragment as a capture, and folate binding protein as a detection;

[0081] Preferably, the time-resolved fluorescent immunochromatography reagent uses (3) any one of antibody 3 or its antigen-binding fragment and folate binding protein as a capture, and the other as a detection; further preferably, the time-resolved fluorescent immunochromatography reagent uses antibody 2 or its antigen-binding fragment as a capture, and folate binding protein as a detection;

[0082] Preferably, the time-resolved fluorescent immunochromatography reagent uses (4) any one of antibody 4 or its antigen-binding fragment and folate binding protein as a capture, and the other as a detection; further preferably, the time-resolved fluorescent immunochromatography reagent uses antibody 2 or its antigen-binding fragment as a capture, and folate binding protein as a detection;

[0083] Preferably, the time-resolved fluorescent immunochromatography reagent uses (5) any one of antibody 5 or its antigen-binding fragment and folate binding protein as a capture, and the other as a detection; further preferably, the time-resolved fluorescent immunochromatography reagent uses antibody 2 or its antigen-binding fragment as a capture, and folate binding protein as a detection;

[0084] Preferably, the time-resolved fluorescent immunochromatography reagent uses (6) any one of antibody 6 or its antigen-binding fragment and folate binding protein as a capture, and the other as a detection; further preferably, the time-resolved fluorescent immunochromatography reagent uses antibody 2 or its antigen-binding fragment as a capture, and folate binding protein as a detection;

[0085] Preferably, the time-resolved fluorescent immunochromatography reagent uses (7) any one of antibody 7 or its antigen-binding fragment and folate binding protein as a capture, and the other as a detection; further preferably, the time-resolved fluorescent immunochromatography reagent uses antibody 2 or its antigen-binding fragment as a capture, and folate binding protein as a detection;

[0086] Preferably, the time-resolved fluorescent immunochromatography reagent uses (8) any one of antibody 8 or its antigen-binding fragment and folate binding protein as a capture, and the other as a detection; further preferably, the time-resolved fluorescent immunochromatography reagent uses antibody 2 or its antigen-binding fragment as a capture, and folate binding protein as a detection;

[0087] Preferably, the time-resolved fluorescent immunochromatography reagent uses (9) any one of antibody 9 or its antigen-binding fragment and folate binding protein as a capture, and uses the other as a detection; further preferably, the time-resolved fluorescent immunochromatography reagent uses antibody 2 or its antigen-binding fragment as a capture, and uses folate binding protein as a detection;

[0088] Preferably, the time-resolved fluorescent immunochromatography reagent uses (10) any one of antibody 10 or its antigen-binding fragment and folate binding protein as a capture, and the other as a detection; further preferably, the time-resolved fluorescent immunochromatography reagent uses antibody 2 or its antigen-binding fragment as a capture, and folate binding protein as a detection;

[0089] Preferably, the folate binding protein used as the detection object is labeled with a detectable marker; such detectable markers are well known to those skilled in the art, including but not limited to colloidal gold, time-resolved fluorescent microspheres, FITC, and the like.

[0090] In a ninth aspect, the present invention provides a method for preparing the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, the method comprising: allowing the transformed host cell as described in the fifth aspect above to express the monoclonal antibody or antigen-binding fragment thereof under conditions suitable for the expression of the monoclonal antibody or antigen-binding fragment thereof, and recovering the expressed monoclonal antibody or antigen-binding fragment thereof from the culture of the host cell.

[0091] In the tenth aspect, the present invention provides a method for detecting the presence or level of folic acid (Folate) in a sample, the method comprising using the monoclonal antibody or its antigen-binding fragment as described in the first aspect above, the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, the expression vector as described in the fourth aspect above, the transformed host cell as described in the fifth aspect above and / or the detection reagent or kit as described in the eighth aspect above.

[0092] In a feasible embodiment, the sample includes but is not limited to serum, plasma, etc. from a subject.

[0093] The method can be used for diagnostic purposes (eg, the sample is a sample from a patient), or for non-diagnostic purposes (eg, the sample is a cell sample rather than a sample from a patient).

[0094] The general method of using monoclonal antibodies or their antigen-binding fragments to detect the presence of target antigens in samples or their levels is well known to those skilled in the art. In certain preferred embodiments, the detection method can use enzyme-linked immunosorbent assay (ELISA), immunochromatographic assay, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, competitive assay and similar detection methods.

[0095] The present invention also relates to a sequence with a similarity of 85% or more to the CDR region. The monoclonal antibody or antigen-binding fragment thereof against folic acid and its binding protein complex comprises:

[0096] (1) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 85% similarity to the amino acid sequences of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of the LCDR1, LCDR2 and LCDR3 have 85% similarity to the amino acid sequences of SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6; or,

[0097] (2) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 85% similarity to the amino acid sequences of SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of the LCDR1, LCDR2 and LCDR3 have 85% similarity to the amino acid sequences of SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16; or,

[0098] (3) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 85% similarity to the amino acid sequences of SEQ ID NO.21, SEQ ID NO.22 and SEQ ID NO.23; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of the LCDR1, LCDR2 and LCDR3 have 85% similarity to the amino acid sequences of SEQ ID NO.24, SEQ ID NO.25 and SEQ ID NO.26; or,

[0099] (4) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 85% similarity to the amino acid sequences of SEQ ID NO.31, SEQ ID NO.32 and SEQ ID NO.33; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of the LCDR1, LCDR2 and LCDR3 have 85% similarity to the amino acid sequences of SEQ ID NO.34, SEQ ID NO.35 and SEQ ID NO.36; or,

[0100] (5) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 85% similarity to the amino acid sequences of SEQ ID NO.41, SEQ ID NO.42 and SEQ ID NO.43; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of the LCDR1, LCDR2 and LCDR3 have 85% similarity to the amino acid sequences of SEQ ID NO.44, SEQ ID NO.45 and SEQ ID NO.46; or,

[0101] (6) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 85% similarity to the amino acid sequences of SEQ ID NO.51, SEQ ID NO.52 and SEQ ID NO.53; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of the LCDR1, LCDR2 and LCDR3 have 85% similarity to the amino acid sequences of SEQ ID NO.54, SEQ ID NO.55 and SEQ ID NO.56; or,

[0102] (7) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 85% similarity to the amino acid sequences of SEQ ID NO.61, SEQ ID NO.62 and SEQ ID NO.63; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of the LCDR1, LCDR2 and LCDR3 have 85% similarity to the amino acid sequences of SEQ ID NO.64, SEQ ID NO.65 and SEQ ID NO.66; or,

[0103] (8) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 85% similarity to the amino acid sequences of SEQ ID NO.71, SEQ ID NO.72 and SEQ ID NO.73; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of the LCDR1, LCDR2 and LCDR3 have 85% similarity to the amino acid sequences of SEQ ID NO.74, SEQ ID NO.75 and SEQ ID NO.76; or,

[0104] (9) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 85% similarity to the amino acid sequences of SEQ ID NO.81, SEQ ID NO.82 and SEQ ID NO.83; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of the LCDR1, LCDR2 and LCDR3 have 85% similarity to the amino acid sequences of SEQ ID NO.84, SEQ ID NO.85 and SEQ ID NO.86; or,

[0105] (10) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 have 85% similarity to the amino acid sequences of SEQ ID NO.91, SEQ ID NO.92 and SEQ ID NO.93; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of the LCDR1, LCDR2 and LCDR3 have 85% similarity to the amino acid sequences of SEQ ID NO.94, SEQ ID NO.95 and SEQ ID NO.96.

[0106] Beneficial effects: The monoclonal antibodies against folic acid and its binding protein complex of the present invention can bind to folic acid and its binding protein complex with high affinity and high specificity (the EC50 of the binding activity reaches the ng / ml level, and the KD reaches 10 -10 M-10 -11 M), achieving high-accuracy and high-sensitivity detection of protein complexes; and, the present invention breaks through the limitations of low sensitivity and poor clinical relevance of traditional competitive detection reagents, and realizes sandwich immunoassay of folic acid (Folate), which has higher specificity and sensitivity, and its clinical compliance rate is above 0.97. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0108] Figure 1 This is a gel image of SDS-PAGE detection of FA mAb1 expressed and purified in Example 4 of the present invention;

[0109] Figure 2 This is a gel image of SDS-PAGE detection of FA mAb2 expressed and purified in Example 4 of the present invention;

[0110] Figure 3 This is a gel image of SDS-PAGE detection of FA mAb3 expressed and purified in Example 4 of the present invention;

[0111] Figure 4 This is a gel image of SDS-PAGE detection of FA mAb4 expressed and purified in Example 4 of the present invention;

[0112] Figure 5 This is a gel image of SDS-PAGE detection of FA mAb5 expressed and purified in Example 4 of the present invention;

[0113] Figure 6 This is a gel image of SDS-PAGE detection of FA mAb6 expressed and purified in Example 4 of the present invention;

[0114] Figure 7 This is a gel image of SDS-PAGE detection of FA mAb7 expressed and purified in Example 4 of the present invention;

[0115] Figure 8 This is a gel image of SDS-PAGE detection of FA mAb8 expressed and purified in Example 4 of the present invention;

[0116] Fig. 9 This is a gel image of SDS-PAGE detection of FA mAb9 expressed and purified in Example 4 of the present invention;

[0117] Fig.10 This is a gel image of SDS-PAGE detection of FA mAb10 expressed and purified in Example 4 of the present invention;

[0118] Figure 1-Figure 10 In the figure, M represents the electrophoretic band of the molecular weight marker, lane 1 shows the electrophoretic band of each monoclonal antibody under reducing conditions, and lane 2 shows the electrophoretic band of each monoclonal antibody under non-reducing conditions.

[0119] Fig.11 This is a graph showing the activity detection results of FA mAb1 expressed and purified in Example 4 of the present invention;

[0120] Fig.12 This is a graph showing the activity detection results of FA mAb2 expressed and purified in Example 4 of the present invention;

[0121] Fig.13 This is a graph showing the activity detection results of FA mAb3 expressed and purified in Example 4 of the present invention;

[0122] Fig.14 This is a graph showing the activity detection results of FA mAb4 expressed and purified in Example 4 of the present invention;

[0123] Fig.15 This is a graph showing the activity detection results of FA mAb5 expressed and purified in Example 4 of the present invention;

[0124] Fig.16 This is a graph showing the activity detection results of FA mAb6 expressed and purified in Example 4 of the present invention;

[0125] Fig.17 This is a graph showing the activity detection results of FA mAb7 expressed and purified in Example 4 of the present invention;

[0126] Fig.18 This is a graph showing the activity detection results of FA mAb8 expressed and purified in Example 4 of the present invention;

[0127] Fig.19 This is a graph showing the activity detection results of FA mAb9 expressed and purified in Example 4 of the present invention;

[0128] Fig. 20 This is a graph showing the activity detection results of FA mAb10 expressed and purified in Example 4 of the present invention;

[0129] Fig.21 The result diagram of the clinical sample correlation between the detection reagent prepared based on the antibody of the present invention and the Roche detection reagent, as detected in Example 7. DETAILED DESCRIPTION

[0130] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising” will be understood to include the stated elements or components but not to exclude other elements or components.

[0131] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0132] In order to make the present invention more easily understood, some technical terms are specifically defined as follows. Unless otherwise clearly defined in other parts of this document, the technical terms used herein have the meanings commonly understood by ordinary technicians in the field to which the present invention belongs.

[0133] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as such variations are appropriate for performing the disclosed methods.

[0134] The term "and / or" should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0135] As used herein, the term "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, that is, including at least one of the numbers or elements in the list, but also including more than one, and optionally, additional unlisted items. Only when terms are clearly indicated to the contrary, such as "only one" or "exactly one" or when "consisting of..." is used in the claims, it will refer to only one number listed or one element of the list.

[0136] The term "percent (%) amino acid sequence identity" or simply "identity" is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the amino acid residues in a reference amino acid sequence, after the amino acid sequences are aligned (and gaps are introduced, if necessary) to obtain the maximum percentage sequence identity, and any conservative substitutions are not considered as part of the sequence identity. Sequence alignments can be performed using various methods in the art to determine the percentage amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignments, including any algorithm required to obtain maximum alignment over the entire length of the compared sequences.

[0137] The term "antibody" refers to any form of antibody having the desired biological activity. Therefore, it is used in the broadest sense, specifically including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies and camelized single domain antibodies.

[0138] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0139] "Antigen binding fragment" refers to an antigen binding fragment of an antibody and antibody analogs, which generally include at least a portion of the antigen binding region or variable region of the parent antibody, such as one or more CDRs. The antibody fragment retains at least some of the binding specificity of the parent antibody. Antigen binding fragments include those selected from Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, diabodies, peptides containing CDRs, etc.

[0140] The "Fab fragment" consists of a light chain and the CH1 and variable regions of a heavy chain.

[0141] The "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domains.

[0142] A "Fab' fragment" contains a light chain and a portion of a heavy chain including a VH domain, a CH1 domain, and a portion of the constant region between the CH1 and CH2 domains, with an interchain disulfide bond formed between the two heavy chains of the two Fab' fragments to form a F(ab')2 molecule.

[0143] A "F(ab')2 fragment" contains two light chains and a portion of two heavy chains including a VH domain, a CH1 domain, and a portion of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, a F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0144] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0145] "Single-chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment comprising the VH and VL domains of an antibody, which domains are contained in a single polypeptide chain. Generally speaking, the scFv polypeptide comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0146] "Diabodies" are small antigen-binding fragments with two antigen-binding sites. The fragments contain a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are allowed to pair with the complementary domains of another chain and form two antigen-binding sites.

[0147] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art.

[0148] The term "high affinity" for IgG antibodies refers to a KD for the antigen of 1.0 × 10 -9 M or lower. For other antibody subtypes, "high affinity" binding may vary.

[0149] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single- or double-stranded form.

[0150] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0151] Example 1: Preparation of monoclonal antibodies against folic acid and its binding protein complex

[0152] (1) Expression and purification of folate binding protein:

[0153] Search UniProt number: P02702 on the UniProt website, find the amino acid sequence of Folate receptor alpha (i.e., folate binding protein), deduce the corresponding gene sequence using software, and synthesize it. The obtained DNA sequence is cloned into the PCDNA3.4 vector, then transformed and plated, cloned, and plasmid extracted.

[0154] The recombinant plasmid was transfected into N293 cells under the following transfection conditions: the mass ratio of PEI / plasmid was 3:1, and the transfection ratio was 2 μg plasmid / 1 ml N293 cells. 5-7 days after transfection, the cell supernatant was collected and purified using a nickel column to obtain a high-purity binding protein. The elution buffer was replaced with PBS by dialysis, and the antibody concentration was determined using the Bradford method.

[0155] (2) Animal immunization:

[0156] The folic acid small molecule and the binding protein were mixed at a molar ratio of 20:1 and allowed to stand for 30 minutes to allow the two to completely bind. The resulting folic acid and its binding protein complex (concentration 0.5 mg / ml) was then used as an immunogen. 30 μg of antigen (i.e., 60 μl) was fully emulsified with an equal amount (60 μl) of Freund's complete adjuvant and then injected intraperitoneally to immunize mice (breed: Balb / c, female, 6-8 weeks old). Reinforcement immunizations were performed at intervals of two weeks for a total of 3 immunizations. A trace amount of tail blood was collected 5-8 days after the last booster immunization, and the serum titer was detected by ELISA. The specific experimental information is shown in Table 1.

[0157] Table 1. Experimental information

[0158]

[0159] The serum titer data detected by ELISA are shown in Table 2.

[0160] Table 2. ELIAS method for testing serum titer data

[0161]

[0162] Mice with a serum titer of 512K were selected to prepare spleen single cell suspension.

[0163] (3) Preparation of spleen single cell suspension:

[0164] The mice were killed by cervical dislocation. A small incision was made in the middle of the left ventral side of the mouse, the skin was torn open, the abdominal wall was exposed, the peritoneum was lifted up on the lower side of the spleen, and the spleen was exposed after being cut open. The spleen was lifted with forceps, and the connective tissue under the spleen was separated with ophthalmic scissors. The spleen was removed. The spleen was placed on a cell filter and gently pressed with a syringe needle to obtain a cell suspension. Another 15 ml sterile test tube was taken, and the cell separation solution Ficoll was first added. The cell suspension just prepared was slowly added to the test tube. The volume ratio of the separation solution to the cell suspension was 1:2. Place it in a centrifuge at 2000 rpm and centrifuge at room temperature for 20 minutes. After centrifugation, the test tube was taken out, and different layers could be seen. The upper non-cellular layer was aspirated and discarded. The single nuclear layer was aspirated in another 15 ml sterile test tube, and centrifuged at 1500 rpm at room temperature for 10 minutes. The supernatant was carefully removed and the cells were resuspended.

[0165] (4) Obtaining single B cells:

[0166] The prepared spleen single cell suspension is used to label the antigen and perform cell screening using a single cell screening platform to obtain antigen-specific B cells.

[0167] Through the above steps, a total of 182 monoclonal antibodies against folic acid and its binding protein complex were obtained, and they were all IgG antibodies after identification, and their names are as shown in Tables 3A and 3B. In the following examples, the light and heavy chain gene sequences of the monoclonal antibodies were detected and expressed, and the transfected cell supernatants were placed in 96-well plates according to the layout of Tables 3A and 3B for binding activity detection.

[0168] Table 3A. 88 antibody supernatant clone numbers

[0169]

[0170] Table 3B. 94 antibody supernatant clone numbers

[0171]

[0172] Example 2: Specific detection of monoclonal antibodies against folate-binding protein complexes

[0173] The supernatant of the cell clones in Example 1 was tested for antigen specificity using ELISA:

[0174] ① Antigen coating: dilute folic acid and its binding protein complex to 2µg / ml, then add 100μl / well to an empty ELISA plate, place the ELISA plate in a 4℃ refrigerator, and incubate overnight;

[0175] ② Blocking: Wash the plate 5 times with a plate washer, tap dry, add blocking solution to the ELISA plate, 150 μl per well, place in a 37°C incubator, block for 1 hour; wash the plate 5 times with a plate washer, tap dry;

[0176] ③ Incubate with primary antibody: add the culture supernatant of 96-well plate to the wells of ELISA plate, 100 μl per well, incubate at 37°C for 1 hour; wash the plate 5 times with a plate washer and dry;

[0177] ④ Incubate with secondary antibody: dilute HRP-labeled goat anti-mouse secondary antibody at 1:10000, then add to the ELISA plate, 100 μl per well, incubate at 37°C for 30 min; wash the plate 5 times with a plate washer and dry;

[0178] ⑤ Color development: add the color development solution to the ELISA plate, 100 μl per well, reaction time: 37℃ / 15min;

[0179] ⑥ Stop and read: add stop solution, volume 100μl / well; detection wavelength setting: detection wavelength 450nm, reference wavelength 620nm, the detection results are shown in Table 4.

[0180] Table 4. Specificity test results

[0181]

[0182] ⑦Data analysis: By comparing the signal strengths, 10 clones with strong reaction signals and specific binding to the folate-binding protein complex were finally selected (i.e., FA mAb1, FA mAb2, FA mAb3, FA mAb4, FA mAb5, FA mAb6, FAmAb7, FA mAb8, FA mAb9 and FA mAb10 clone numbers are 2M1B5, 1M1C4, 1M1A11, 2M2E5, 1M2B2, 1M4F4, 2M3F6, 2M4D3, 1M4F3 and 2M4G12, respectively) for subsequent amplification, expression and purification.

[0183] Example 3: Obtaining the amino acid sequences of the light and heavy chains of monoclonal antibodies against the folate-binding protein complex

[0184] (1) cDNA preparation:

[0185] RNA was extracted from the cells corresponding to the two clones determined in Example 2, and reverse transcription was performed using the RNA as a template to obtain cDNA;

[0186] (2) Acquisition of light and heavy chain genes:

[0187] Amplify using specific primers, clone the amplified DNA sequences into the PCDNA3.4 vector, transform the plate, pick the clones, and extract the plasmids. Send the plasmids for sequencing to obtain the gene sequences of 10 monoclonal antibodies, and deduce the heavy and light chain amino acid sequences of the 10 monoclonal antibodies. The heavy chain amino acid sequences are shown in SEQ ID NO.9, 19, 29, 39, 49, 59, 69, 79, 89, and 99, respectively, and the light chain amino acid sequences are shown in SEQ ID NO.10, 20, 30, 40, 50, 60, 70, 80, 90, and 100, respectively.

[0188] FA mAb1:

[0189] Heavy chain full length (SEQ ID NO.9)

[0190] EVHLQQSGPELVKPGASMKISCKTSGYTFTDYNMYWVKQSHGKSLEWIGGINPNNGRTNYNQKFKGKAILTVDKSSSAAYLELRSLTSEDSAVYFCARSGYYHYFDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0191] Full-length light chain (SEQ ID NO.10)

[0192] DIVMTQSPSSLSVSAGEKVTMSCKSSSSVSSSYLAWYQQRPGQPPKLLIYSTSTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCHQWSSYPPTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0193] FA mAb2:

[0194] Full-length heavy chain (SEQ ID NO.19)

[0195] EVLLQQSGPELVKSGASVKIPCKASGYTFTECTMDWVKQSHGKSLEWIGDINPNNGGTLYNQKFEGKATLTVDKSPSTAYMGLRSLTSEDSAVYYCARSPIYYGYYYYAMDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0196] Full - length light chain (SEQ ID NO.20)

[0197] QIVLTQSPAILSASPGEKVTLTCSASQSLLNSGNQKNYLYWYQQKPGSSPKLWIYGASNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCQNDHSYSLTFGAGTKLELIRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0198] FA mAb3

[0199] Full - length heavy chain (SEQ ID NO.29)

[0200] QVQLKESGPGLVAPSQSLSITCTVSGYTFTDHNIYWVRQPPGKGLEWLGMINPNNGGTDRNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARSVTGTYYFDYWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0201] Full-length light chain (SEQ ID NO.30)

[0202] DIQMTQSPSSLSTSLGGKVTITCKASSSVSSSYIAWYQHKPGKGPRLLIHSTSTLQPDIPSRFSGSGSGRDYSFSISNLEPEDIATYYCHQWSSYPPTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0203] FA mAb4

[0204] Full-length heavy chain (SEQ ID NO.39)

[0205] QVQLKESGPGLVAPSQSLSITCTVSGFAFNQYDIYWVRQPPGKGLEWLGMISSGGGNTDRNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARQRSSYGWYFYAMDYWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0206] Full-length light chain (SEQ ID NO.40)

[0207] DIQMTQSPSSLSTSLGGKVTITCKASRSLLNSENQKNYIAWYQHKPGKGPRLLIHAASTLQPDIPSRFSGSGSGRDYSFSISNLEPEDIATYYCQNDHSYPITFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0208] FA mAb5

[0209] Full-length heavy chain (SEQ ID NO.49)

[0210] EVLLQQSGPELVKPGASVKISCKASGYAFTSSWMDWVKQSQGKSLEWIGDIHPKSGNTVYNQKFKGKATLTLDKSSSTAYMELRRLTSEDTAVYYCARDGDFGYWYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0211] Full-length light chain (SEQ ID NO.50)

[0212] DIQMTQTTSSPSASLGDRVTISCRASKSLLHSNGITFLNWFQQKPDGTVKLLIYQMSKLHSGVPSRFSGSGSGADYSLTISNLEQEDIATYFCAQNLELPWSFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0213] FA mAb6

[0214] Full-length heavy chain (SEQ ID NO.59)

[0215] EVLLQQSGPELVKPGASVKISCKASGYAFTSSWMDWVKQSQGKSLEWIGDIHPKSGNTVYNQKFKGKATLTLDKSSSTAYMELRRLTSEDTAVYYCARDGDYGYYYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0216] Full-length light chain (SEQ ID NO.60)

[0217] DIQMTQTTSSPSASLGDRVTISCRASKSLLHSNGITYLNWFQQKPDGTVKLLIYQMSKLHSGVPSRFSGSGSGADYSLTISNLEQEDIATYFCAQNLELPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0218] FA mAb7:

[0219] Full-length heavy chain (SEQ ID NO.69)

[0220] EVQLVESGGGLVKPGGSLKLSCAASGYSFTDYNMSWVRQTPEKRLEWVAYINPNNGRTYYSDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARSGDNYYFDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0221] Full - length light chain (SEQ ID NO.70)

[0222] DIVMTQSPSSLSVSAREKVTMRCKSSSSVSSSYLAWYQQKPGQPPKLLIYSTSTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAFYYCHQWSSYPPTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0223] FA mAb8

[0224] Full - length heavy chain (SEQ ID NO.79)

[0225] EVLLQQSGPELVKSGTSVKISCKASGFAFNNYDMDWVKQSHGKSLEWIGDISSGGGNTIYNPKFRGKATLTVDKSSGTAYMELRSLTSEDTAVYYCARQRSSYGYYFYAMDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0226] Full - length light chain (SEQ ID NO.80)

[0227] QIVLTQSSTIMSASPGERVTLTCSASRSLLNSENQKNYLYWYQQKPGSSPKLWIYGASNLASGVPARFSGSGSGTSYSLTISTMEAEDAASYFCQNDHSYPLTFGGGTKLEINRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0228] FA mAb9

[0229] Full - length heavy chain (SEQ ID NO.89)

[0230] QVQLQQSAAELARPGASVKMSCKASGYSFTAYTMHWVKQRPGQGLEWIGYINPNNGGTVYNQNLKDKTTLTADKSSSTAYMQLRSLTSEDSAVYYCSRNPIYYGNYYCAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0231] Full - length light chain (SEQ ID NO.90)

[0232] DIKMTQSPSSMYASLGERVTITCKASQSLLNSGNQKNYLSWFQQKPGKPPKTLIYGASRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCQNDHSYPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0233] FA mAb10:

[0234] Full - length heavy chain (SEQ ID NO.99)

[0235] EVQLQQSGPELVKPGASVKISCKTSGYTFTSYTIHWVKQSHGKSLEWIGGINPSSGYTTYNQKFKGKATLTVDKSSSTAYMEVRSLTSEDSAVYYCASNTLYYYGSSQPHFY SMDYWGQGTSVTASSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIV PRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEK TISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0236] Light chain full length (SEQ ID NO.100)

[0237] DIVMTQSPSFLSVSAGEKVTMSCKSSQGINSYLAWYQQKPGQPPKLLIYRANRTRESGVPDRFRGSGSGTDFTLTINGVQAEDLAVYYCLQYDEFPLTFGAGTKLEL KRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0238] The heavy chain variable regions (FV) and light chain variable regions (FV) of the 10 monoclonal antibodies were then analyzed based on the sequencing results, and finally the amino acid sequences of their heavy chain variable regions and light chain variable regions were obtained, as shown below, respectively, where the underlined regions are their CDR regions.

[0239] FA mAb1:

[0240] Heavy chain FV (SEQ ID NO.7)

[0241] EVHLQQSGPELVKPGASMKISCKTSGYTFTDYNMYWVKQSHGKSLEWIGGINPNNGRTNYNQKFKGKAILTVDKSSSAAYLELRSLTSEDSAVYFCARSGYYHYFDYWGQGTSVTVSS

[0242] Light chain FV (SEQ ID NO.8)

[0243] DIVMTQSPSSLSVSAGEKVTMSCKSSSSVSSSYLAWYQQRPGQPPKLLIYSTSTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCHQWSSYPPTFGAGTKLELK

[0244] That is, the heavy chain variable region has HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively; the light chain variable region has LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively;

[0245] SEQ ID NO.1: GYTFTDYN

[0246] SEQ ID NO.2: INPNNGRT

[0247] SEQ ID NO.3: ARSGYYHYFDY

[0248] SEQ ID NO.4:SSVSSSY

[0249] SEQ ID NO.5: STS

[0250] SEQ ID NO.6: HQWSSYPPT

[0251] FA mAb2:

[0252] Heavy chain FV (SEQ ID NO.17)

[0253] EVLLQQSGPELVKSGASVKIPCKASGYTFTECTMDWVKQSHGKSLEWIGDINPNNNGGTLYNQKFEGKATLTVDKSPSTAYMGLRSLTSEDSAVYYCARSPIYYGYYYYAMDYWGQGTTLTVSS

[0254] Light chain FV (SEQ ID NO.18)

[0255] QIVLTQSPAILSASPGEKVTLTCSASQSLLNSGNQKNYLYWYQQKPGSSPKLWIYGASNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCQNDHSYSLTFGAGTKLELI

[0256] That is, the heavy chain variable region has HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13, respectively; the light chain variable region has LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16, respectively;

[0257] SEQ ID NO.11: GYTFTECT

[0258] SEQ ID NO.12: INPNNGGT

[0259] SEQ ID NO.13: ARSPIYYGYYYYAMDY

[0260] SEQ ID NO.14: QSLLNSGNQKNY

[0261] SEQ ID NO.15: GAS

[0262] SEQ ID NO.16: QNDHSYSLT

[0263] FA mAb3:

[0264] Heavy chain FV (SEQ ID NO.27)

[0265] QVQLKESGPGLVAPSQSLSITCTVSGYTFTDHNIYWVRQPPGKGLEWLGMINPNNGGTDRNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARSVTGTYYFDYWGAGTTVTVSS

[0266] Light chain FV (SEQ ID NO.28)

[0267] DIQMTQSPSSSLSTSLGGKVTITCKASSSVSSSYIAWYQHKPGKGPRLLIHSTSTLQPDIPSRFSGSGSGRDYSFSISNLEPEDIATYYCHQWSSYPPTFGGGTKLEIK

[0268] That is, the heavy chain variable region has HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO.21, SEQ ID NO.22 and SEQ ID NO.23, respectively; the light chain variable region has LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO.24, SEQ ID NO.25 and SEQ ID NO.26, respectively;

[0269] SEQ ID NO.21: GYTFTDHN

[0270] SEQ ID NO.22: INPNNGGT

[0271] SEQ ID NO.23: ARSVTGTYYFDY

[0272] SEQ ID NO.24:SSVSSSY

[0273] SEQ ID NO.25: STS

[0274] SEQ ID NO.26: HQWSSYPPT

[0275] FA mAb4:

[0276] Heavy chain FV (SEQ ID NO.37)

[0277] QVQLKESGPGLVAPSQSLSITCTVSGFAFNQYDIYWVRQPPGKGLEWLGMISSGGGNTDRNSALKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARQRSSYGWYFYAMDYWGAGTTVTVSS

[0278] Light chain FV (SEQ ID NO.38)

[0279] DIQMTQSPSSSLSTSLGGKVTITCKASRSLLNSENQKNYIAWYQHKPGKGPRLLIHAASTLQPDIPSRFSGSGSGRDYSFSISNLEPEDIATYYCQNDHSYPITFGGGTKLEIK

[0280] That is, the heavy chain variable region has HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO.31, SEQ ID NO.32 and SEQ ID NO.33, respectively; the light chain variable region has LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO.34, SEQ ID NO.35 and SEQ ID NO.36, respectively;

[0281] SEQ ID NO.31: GFAFNQYD

[0282] SEQ ID NO.32: ISSGGGNT

[0283] SEQ ID NO.33:ARQRSSYGWYFYAMDY

[0284] SEQ ID NO.34: RSLLNSENQKNY

[0285] SEQ ID NO.35: AAS

[0286] SEQ ID NO.36: QNDHSYPIT

[0287] FA mAb5:

[0288] Heavy chain FV (SEQ ID NO.47)

[0289] EVLLQQSGPELVKPGASVKISCKASGYAFTSSWMDWVKQSQGKSLEWIGDIHPKSGNTVYNQKFKGKATLTLDKSSSTAYMELRRLTSEDTAVYYCARDGDFGYWYAMDYWGQGTSVTVSS

[0290] Light chain FV (SEQ ID NO.48)

[0291] DIQMTQTTSSPSASLGDRVTISCRASKSLLHSNGITFLNWFQQKPDGTVKLLIYQMSKLHSGVPSRFSGSGSGADYSLTISNLEQEDIATYFCAQNLELPWSFGGGTKLEIK

[0292] That is, the heavy chain variable region has HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO.41, SEQ ID NO.42 and SEQ ID NO.43, respectively; the light chain variable region has LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO.44, SEQ ID NO.45 and SEQ ID NO.46, respectively;

[0293] SEQ ID NO.41: GYAFTSSW

[0294] SEQ ID NO.42: IHPKSGNT

[0295] SEQ ID NO.43: ARDGDFGYWYAMDY

[0296] SEQ ID NO.44: KSLLHSNGITF

[0297] SEQ ID NO.45: QMS

[0298] SEQ ID NO.46: AQNLELPWS

[0299] FA mAb6:

[0300] Heavy chain FV (SEQ ID NO.57)

[0301] EVLLQQSGPELVKPGASVKISCKASGYAFTSSWMDWVKQSQGKSLEWIGDIHPKSGNTVYNQKFKGKATLTLDKSSSTAYMELRRLTSEDTAVYYCARDGDYGYYYAMDYWGQGTSVTVSS

[0302] Light chain FV (SEQ ID NO.58)

[0303] DIQMTQTTSSPSASLGDRVTISCRASKSLLHSNGITYLNWFQQKPDGTVKLLIYQMSKLHSGVPSRFSGSGSGADYSLTISNLEQEDIATYFCAQNLELPWTTFGGGTKLEIK

[0304] That is, the heavy chain variable region has HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO.51, SEQ ID NO.52 and SEQ ID NO.53, respectively; the light chain variable region has LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO.54, SEQ ID NO.55 and SEQ ID NO.56, respectively;

[0305] SEQ ID NO.51: GYAFTSSW

[0306] SEQ ID NO.52: IHPKSGNT

[0307] SEQ ID NO.53: ARDGDYGYYYAMDY

[0308] SEQ ID NO.54: KSLLHSNGITY

[0309] SEQ ID NO.55: QMS

[0310] SEQ ID NO.56: AQNLELPWT

[0311] FA mAb7:

[0312] Heavy chain FV (SEQ ID NO.67)

[0313] EVQLVESGGGLVKPGGSLKLSCAASGYSFTDYNMSWVRQTPEKRLEWVAYINNPNNGRTYYSDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARSGDNYYFDYWGQGTSVTVSS

[0314] Light chain FV (SEQ ID NO.68)

[0315] DIVMTQSPSSSVSAREKVTMRCKSSSSVSSSYLAWYQQKPGQPPKLLIYSTSTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAFYYCHQWSSYPPTFGAGTKLELK

[0316] That is, the heavy chain variable region has HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO.61, SEQ ID NO.62 and SEQ ID NO.63, respectively; the light chain variable region has LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO.64, SEQ ID NO.65 and SEQ ID NO.66, respectively;

[0317] SEQ ID NO.61: GYSFTDYN

[0318] SEQ ID NO.62: INPNNGRT

[0319] SEQ ID NO.63: ARSGDNYYFDY

[0320] SEQ ID NO.64:SSVSSSY

[0321] SEQ ID NO.65: STS

[0322] SEQ ID NO.66: HQWSSYPPT

[0323] FA mAb8:

[0324] Heavy chain FV (SEQ ID NO.77)

[0325] EVLLQQSGPELVKSGTSVKISCKASGFAFNNYDMDWVKQSHGKSLEWIGDISSGGGNTIYNPKFRGKATLTVDKSSGTAYMELRSLTSEDTAVYYCARQRSSYGYYFYAMDYWGQGTTLTVSS

[0326] Light chain FV (SEQ ID NO.78)

[0327] QIVLTQSSTIMSASPGERVTLTCSASRSLLNSENQKNYLYWYQQKPGSSPKLWIYGASNLASGVPARFSGSGSGTSYSLTISTMEAEDAASYFCQNDHSYPLTFGGGTKLEIN

[0328] That is, the heavy chain variable region has HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO.71, SEQ ID NO.72 and SEQ ID NO.73, respectively; the light chain variable region has LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO.74, SEQ ID NO.75 and SEQ ID NO.76, respectively;

[0329] SEQ ID NO.71: GFAFNNYD

[0330] SEQ ID NO.72: ISSGGGNT

[0331] SEQ ID NO.73:ARQRSSYGYYFYAMDY

[0332] SEQ ID NO.74: RSLLNSENQKNY

[0333] SEQ ID NO.75: GAS

[0334] SEQ ID NO.76: QNDHSYPLT

[0335] FA mAb9:

[0336] Heavy chain FV (SEQ ID NO.87)

[0337] QVQLQQSAAELARPGASVKMSCKASGYSFTAYTMHWVKQRPGQGLEWIGYINPNNGGTVYNQNLKDKTTLTADKSSSTAYMQLRSLTSEDSAVYYCSRNPIYYGNYYCAMDYWGQGTSVTVSS

[0338] Light chain FV (SEQ ID NO.88)

[0339] DIKMTQSPSSMYASLGERVTITCKASQSLLNSGNQKNYLSWFQQKPGKPPKTLIYGASRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCQNDHSYPLTFGAGTKLELK

[0340] That is, the heavy chain variable region has HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO.81, SEQ ID NO.82 and SEQ ID NO.83, respectively; the light chain variable region has LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO.84, SEQ ID NO.85 and SEQ ID NO.86, respectively;

[0341] SEQ ID NO.81: GYSFTAYT

[0342] SEQ ID NO.82: INPNNGGT

[0343] SEQ ID NO.83: SRNPIYYGNYYCAMDY

[0344] SEQ ID NO.84: QSLLNSGNQKNY

[0345] SEQ ID NO.85: GAS

[0346] SEQ ID NO.86: QNDHSYPLT

[0347] FA mAb10:

[0348] Heavy chain FV (SEQ ID NO.97)

[0349] EVQLQQSGPELVKPGASVKISCKTSGYTFTSYTIHWVKQSHGKSLEWIGGINPSSGYTTYNQKFKGKATLTVDKSSSTAYMEVRSLTSEDSAVYYCASNTLYYYGSSQPHFYSMDYWGQGTSVTASS

[0350] Light chain FV (SEQ ID NO.98)

[0351] DIVMTQSPSFLSVSAGEKVTMSCKSSQGINSYLAWYQQKPGQPPKLLIYRANRTRESGVPDRFRGSGSGTDFTLTINGVQAEDLAVYYCLQYDEFPLTFGAGTKLELK

[0352] That is, the heavy chain variable region has HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO.91, SEQ ID NO.92 and SEQ ID NO.93, respectively; the light chain variable region has LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO.94, SEQ ID NO.95 and SEQ ID NO.96, respectively;

[0353] SEQ ID NO.91: GYTFTSYT

[0354] SEQ ID NO.92: INPSGYT

[0355] SEQ ID NO.93:ASNTLYYYGSSQPHFYSMDY

[0356] SEQ ID NO.94: QGINSY

[0357] SEQ ID NO.95: RAN

[0358] SEQ ID NO.96: LQYDEFPLT

[0359] Example 4: Expression and purification of monoclonal antibodies against folate and its binding protein complex

[0360] (1) Cultivate N293 suspension cells until the density reaches (1-3)×10 6 When the cell count is 10 cells / ml and the survival rate is >80-90%, cell transfection is performed;

[0361] (2) The light and heavy chain genes of the 10 monoclonal antibodies obtained in Example 3 were respectively constructed into the pcDNA3.4 plasmid; then, the recombinant plasmids containing the antibody light and heavy chain genes were transfected into N293 cells under the following transfection conditions: the mass ratio of PEI / plasmid was 3:1, and the transfection ratio was 2 μg plasmid / 1 ml N293 cells;

[0362] (3) 5-7 days after transfection, collect the cell supernatant and perform affinity purification with Protein A resin to obtain high-purity monoclonal antibodies. Replace the elution buffer with PBS by dialysis, and measure the antibody concentration using Nanodrop (microspectrophotometer).

[0363] Next, the purity of the antibody was tested by SDS-PAGE (polyacrylamide gel electrophoresis), as follows:

[0364] SDS-PAGE detection:

[0365] Take 2μg of the antibody to be tested, add an appropriate amount of SDS-PAGE protein loading buffer to make the total volume 20μl; prepare reducing SDS-PAGE samples and non-reducing SDS-PAGE samples at the same time, and then load them; first, electrophoresis at 80V for 30min, and then electrophoresis at 120V until the bands are clearly separated; remove the PAGE gel after electrophoresis, stain with Coomassie Brilliant Blue, remove the staining solution after 15min, rinse with clean water, and then add decolorizing solution for decolorization until clear bands are seen. The results are as follows Figure 1-Figure 10 As shown, Figure 1-Figure 10 The results showed that under reducing conditions, all 10 monoclonal antibodies had two electrophoretic bands, and the corresponding molecular weights of the two electrophoretic bands were consistent with the sizes of their respective light and heavy chains; under non-reducing conditions, all monoclonal antibodies had a single band, and the corresponding molecular weights of the electrophoretic bands were consistent with the sizes of their respective complete antibodies; these results indicated that the expression of these 10 monoclonal antibodies was accurate.

[0366] In addition, all electrophoresis bands had clear edges and no foreign bands, indicating that the monoclonal antibodies obtained by the above expression and purification steps had high purity.

[0367] Example 5: Antigen Binding Activity and Affinity Detection of Monoclonal Antibodies Against Folic Acid

[0368] In this example, the antigen activity and affinity of the 10 monoclonal antibodies FA mAb1, FAmAb10 expressed and purified in Example 4 were tested respectively.

[0369] ① Antigen coating: dilute the antigen (i.e., the aforementioned folate-binding protein complex) to an appropriate concentration, and then add it to an empty ELISA plate at a volume of 100 μl / well; place the ELISA plate in a 4°C refrigerator and incubate overnight;

[0370] ② Blocking: Wash the plate 5 times with a plate washer, tap dry, add blocking solution to the ELISA plate, 150 μl per well, place in a 37°C incubator, block for 1 hour; wash the plate 5 times with a plate washer, tap dry;

[0371] ③ Incubate the primary antibody: dilute the antibody to be tested to a suitable concentration gradient with 1×PBS; add the diluted antibody to be tested to the ELISA plate, 100 μl per well, reaction time: 37℃ / 1h; wash the plate 5 times with a plate washer and dry;

[0372] ④ Incubate the secondary antibody: dilute the enzyme-labeled secondary antibody in an appropriate ratio, and then add it to the ELISA plate, 100 μl per well, reaction time: 37°C / 30 min; wash the plate 5 times with a plate washer and dry;

[0373] ⑤ Color development: add the color development solution to the ELISA plate, 100 μl per well, reaction time: 37℃ / 15min;

[0374] ⑥ Stop and read: add stop solution, volume 100μl / well; detection wavelength setting: detection wavelength 450nm, reference wavelength 620nm, export the detection results into Excel table.

[0375] ⑦Data analysis: Use software to analyze and plot data, fit EC50, and derive its KD.

[0376] The test data are shown in Table 5.

[0377] Table 5. OD450 values ​​of the tested antibodies at various concentrations

[0378]

[0379] According to Table 5, the binding activity curve of antibody and antigen was prepared. The results are as follows Figure 11-Figure 20 As shown, the data in Table 5 were fitted with a four-parameter curve using the "ELISAcalc" software to obtain the EC50 values ​​of the 10 antibodies and the KD values ​​derived therefrom. The data are shown in Table 6.

[0380] Table 6. EC50 and KD values ​​of 10 antibodies

[0381]

[0382] Figure 11-Figure 20 The results show that the EC50 of the 10 monoclonal antibodies FA mAb1-FA mAb10 binding to the antigen all reached the ng / ml level, and the KD all reached 10 -10 M-10 -11 As mentioned above, for IgG antibodies, it is generally believed in the art that when the KD value of binding to the antigen reaches 10 -9 M is considered to have high affinity, and the KD values ​​of the 10 monoclonal antibodies of the present invention for binding to the antigen all reached 10 -10 M-10 -11 M is 10-100 times the above-defined value, from which it can be concluded that the monoclonal antibody of the present invention has extremely strong antigen binding activity and affinity.

[0383] Example 6: Preparation of a time-resolved fluorescent immunochromatographic reagent based on antibodies for detecting folic acid and its binding protein complex

[0384] (1) Preparation of fluorescent microspheres-folic acid and its binding protein complex

[0385] ① Pretreatment of time-resolved fluorescent microspheres

[0386] Take 25 μl of time-resolved fluorescent microspheres, add 400 μl of PB buffer solution at pH 6.0, mix well, centrifuge at 14000 rpm, 10°C for 15 min, remove the supernatant, and resuspend the precipitate in 400 μl of PB buffer solution at pH 6.0; prepare 2.5 mg / ml carbodiimide and succinimide, add 2.5 μl each, react at room temperature for 30 min, centrifuge at 14000 rpm, 10°C for 15 min, and resuspend the precipitate in 200 μl of PB solution at pH 6.0 to obtain an activated microsphere solution;

[0387] ② Microsphere-coupled folate binding protein

[0388] 10-50 μg of folate binding protein was added to the above-mentioned time-resolved fluorescent microsphere solution, mixed, reacted at room temperature for 3 h, blocked with 1-5% BSA at room temperature for 0.5 h, centrifuged at 14000 rpm, 10°C for 15 min, the supernatant was removed, and the precipitate was resuspended with 50 μl of preservation solution to obtain the fluorescent microsphere-folate and its binding protein complex; the preservation solution contained 10-100 mM Tris buffer at pH 7.5-8.5, 1-5% sucrose, 1-5% trehalose, and 1-5% BSA.

[0389] (2) Gold spraying

[0390] The final concentration of the fluorescent microsphere-folic acid and its binding protein complex was diluted to 0.5-2 mg / ml, and the gold spraying volume was set to 1-4 μl / cm, and then the sprayed labeling pad was placed in a 37°C oven to dry for 1-4 hours, and then the labeling pad was placed in an aluminum foil bag pre-added with desiccant at a humidity not higher than 36%, and sealed for storage;

[0391] (3) Coating quality control line C, detection line T

[0392] ① Preparation of coating solution for quality control line C and detection line T: Take goat anti-chicken antibody (manufacturer: Jiangsu Huakong Biotechnology Co., Ltd., item number: 2-D5021P), dilute it with 10-100mM PBS buffer at pH 7.4 to make an antibody solution of 0.5-1.0mg / ml; take the antibody for detecting folic acid and its binding protein complex of the present invention, dilute it with 10-100mM PBS buffer at pH 7.4 to make an antibody solution of 0.5-1.0mg / ml;

[0393] ② Coating: Coat the control line C and the detection line T coating solution to the control line and the detection line of the nitrocellulose membrane respectively, with the interval between the C and T lines being 5-8mm, and the coating amount being 0.8-1.5μl / cm2; Place the coated PVC base plate in a 37℃ oven for 12-24h, and then place the PVC base plate in an aluminum foil bag pre-added with desiccant at a humidity not higher than 36% and seal it for future use;

[0394] (4) Pretreatment of sample pad

[0395] Clean the drying net, soak the sample pad with the sample pad pretreatment solution, and then drain it by rotating until no water drops. Then, dry it in a 37°C oven for 4-6 hours, and then seal it in an aluminum foil bag with desiccant added in advance at a humidity not higher than 36% for future use.

[0396] The sample pad pretreatment solution contains 10-100 mM Tris buffer at pH 7.5-8.5, 0.1-0.5% PVP 10, 0.1-0.5% PEG20000, 0.1-0.5% Tween20 and 0.1-0.5% casein sodium;

[0397] (5) Assembly and cutting of membrane materials

[0398] At a humidity not higher than 36%, take out the absorbent pad, the treated sample pad and the marking pad, cut them according to the size of (15-20mm)*300mm, take out the coated PVC base plate, and assemble them according to the following process: first stick the marking pad on the PVC base plate so that the marking pad presses 1-2mm against the nitrocellulose membrane, then stick the sample pad so that the sample pad part presses 3-5mm against the marking pad, and finally stick the absorbent pad so that the absorbent pad presses 1-2mm against the nitrocellulose membrane; cut the assembled PVC base plate into 3-5mm time-resolved fluorescent immunochromatographic test strips for detecting HBP.

[0399] (6) Test strip testing

[0400] The clinical samples were diluted 10 times with sample diluent, 75 μl was added, reacted for 15 min, detected with fluorescent immunoassay analyzer, and the data were processed.

[0401] The antibody pairing methods of the test strips are as follows (select the four strains with the best affinity in ELISA test results for pairing):

[0402] ① Time-resolved fluorescence immunochromatography reagent 4: folate binding protein (used to prepare microsphere-folate and its binding protein complex) + FA mAb4 full-length antibody (used for T-line coating);

[0403] ② Time-resolved fluorescence immunochromatography reagent 5: folate binding protein (used to prepare microsphere-folate and its binding protein complex) + FA mAb5 full-length antibody (used for T-line coating);

[0404] ③ Time-resolved fluorescence immunochromatography reagent 6: folate binding protein (used to prepare microsphere-folate and its binding protein complex) + FA mAb6 full-length antibody (used for T-line coating);

[0405] ④ Time-resolved fluorescent immunochromatography reagent 9: folate binding protein (used to prepare microsphere-folate and its binding protein complex) + FA mAb9 full-length antibody (used for T-line coating).

[0406] Example 7: Detection performance of time-resolved fluorescent immunochromatographic reagent based on anti-folate-binding protein complex antibodies

[0407] In this example, the four time-resolved fluorescent immunochromatographic reagents prepared in Example 6 were used to detect folic acid in clinical serum samples, and compared with Roche's detection reagents.

[0408] (1) Testing clinical serum samples

[0409] Specifically, for the time-resolved fluorescent immunochromatography reagent prepared in Example 6, the clinical serum sample was diluted with a sample diluent (whose composition is: 10mM PBS + 0.05% DTT + 0.5% Tween-20 + 0.1% proclin300, pH7.0), the sample dilution ratio was 1:15 (serum: diluent, v / v), reacted at room temperature for 10 minutes, and then 80µl of the sample was added. After 15 minutes, the dry fluorescent immunoassay was used for detection, the detection signal value was read, and the data was processed; for the Roche detection kit, the operation was carried out according to the instructions, the serum sample was diluted, and then detected to obtain the folic acid concentration data. Then, the folic acid signal value detected by the test strip was compared with the folic acid concentration value detected by the Roche reagent to prepare a clinical correlation curve.

[0410] The specific results are as follows:

[0411] Tables 7 to 10 are the test results of the time-resolved fluorescence immunochromatography reagent, respectively. The first column in the table represents the concentration of clinical samples detected by Roche reagent (unit: ng / ml), and the second column represents the signal value of the corresponding clinical sample detected by the time-resolved fluorescence immunochromatography reagent of the present invention.

[0412] Table 7. Detection results of time-resolved fluorescence immunochromatography reagent 4

[0413]

[0414] Table 8. Detection results of time-resolved fluorescence immunochromatography reagent 5

[0415]

[0416] Table 9. Detection results of time-resolved fluorescence immunochromatography reagent 6

[0417]

[0418] Table 10. Detection results of time-resolved fluorescence immunochromatography reagent 9

[0419]

[0420] According to the results in Tables 7 to 10, the clinical relevance curves of the time-resolved fluorescence immunochromatographic reagents 4, 5, 6, and 9 for detecting folic acid in serum samples (i.e., the coincidence rate between the test results of the time-resolved fluorescence immunochromatographic reagents 4, 5, 6, and 9 and the Roche test results) were prepared respectively. The results are shown in the figure. Figure 3 These results show that the time-resolved fluorescent immunochromatographic reagent of the present invention has a clinical relevance for detecting folic acid in serum samples. 2 All>0.97.

[0421] The above results show that the folic acid time-resolved fluorescent immunochromatographic reagent prepared based on the antibody of the present invention and the folic acid binding protein has a high consistency rate (R 2 above 0.97), thus having good clinical relevance.

[0422] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof for detecting folic acid and its binding protein complex, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises: The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO.51, SEQ ID NO.52 and SEQ ID NO.53, respectively; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO.54, SEQ ID NO.55 and SEQ ID NO.56, respectively.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises: The heavy chain variable region has the amino acid sequence shown in SEQ ID NO.57; and the light chain variable region has the amino acid sequence shown in SEQ ID NO.

58.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises: The heavy chain, whose amino acid sequence is shown in SEQ ID NO.59; the light chain, whose amino acid sequence is shown in SEQ ID NO.

60.

4. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: The monoclonal antibody or antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, Fv, and single-chain antibody.

5. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: It is a chimeric antibody.

6. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: It is a bispecific or multispecific antibody.

7. A polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. A nucleic acid construct comprising the polynucleotide according to claim 7, and at least one expression control element operably linked to the polynucleotide.

9. A recombinant vector comprising the polynucleotide according to claim 7, or the nucleic acid construct according to claim 8. 10 . A transformed host cell comprising the polynucleotide of claim 7 , the nucleic acid construct of claim 8 , or the recombinant vector of claim 9 .

11. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the polynucleotide according to claim 7, the nucleic acid construct according to claim 8, the recombinant vector according to claim 9 and / or the transformed host cell according to claim 10 in the preparation of a detection reagent or kit for detecting folic acid.

12. A detection reagent for detecting folic acid, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the polynucleotide according to claim 7, the nucleic acid construct according to claim 8, the recombinant vector according to claim 9 and / or the transformed host cell according to claim 10.

13. The detection reagent according to claim 12, characterized in that The reagent is a time-resolved fluorescent immunochromatography reagent, and the monoclonal antibody or its antigen-binding fragment and the folate binding protein described in claim 1 are used as the capture object and the detection object respectively.

14. A method for preparing the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the method comprising: Under conditions suitable for the expression of the monoclonal antibody or its antigen-binding fragment, the transformed host cell according to claim 10 is allowed to express the monoclonal antibody or its antigen-binding fragment, and the expressed monoclonal antibody or its antigen-binding fragment is recovered from the culture of the host cell.

Images