KR20180041466A - Antibody specifically binding to core fucosylated alpha-fetoprotein and use thereof - Google Patents

Abstract

The present invention relates to an antibody which specifically binds to core-fucosylated alpha-fetoprotein L3 and uses thereof, and more particularly to an antibody and an antibody-based quantification method of core-fucosylated alpha-fetoprotein for early diagnosis of liver cancer. The antibody of the present invention can be selectively bound to AFP-L3 by distinguishing AFP-L3 from AFP which is not core-fucosylated.

Description

Antibodies specifically binding to core-fucosylated alpha-fetoprotein and uses thereof < RTI ID = 0.0 >

The present invention relates to an antibody that specifically binds to core-fucosylated alpha-fetoprotein (AFP) and its use, and more particularly to an antibody that binds to core-fucosylated alpha-fetoprotein Antibody and antibody-based assays.

It is the early screening of cancer to reduce the cancer burden by minimizing the cancer occurrence and the cancer death through comprehensive cancer management. Alpha-Fetoprotein (AFP) for the screening and monitoring of liver cancer to develop methods for rapidly diagnosing cancer in a large number of people in the concept of screening and monitoring of cancer Monoclonal Development of antibodies is required.

AFP is a glycoprotein that is a tumor marker for hepatocellular carcinoma (HCC). AFP is highly structurally homologous to albumin and is produced in the liver and yolk of the embryo, reaching its peak at 13-14 weeks of gestation. It begins to decrease after birth and decreases to sex inches (<10ug / L) after 12 months. Increased AFP in the maternal blood and amniotic fluid of pregnant women may be used as an indicator of congenital abnormalities such as brain abscess, rupture of spine, and hydrocephalus.

Unlike most tumor markers, AFP is very useful for screening and is being used to screen high risk patients for hepatocellular carcinoma in areas with high prevalence of hepatocellular carcinoma such as Korea, China, Japan, Alaska and Africa. A decrease in tumor markers indicates successful treatment, while an increase in tumor markers indicates recurrence or metastasis, so tumor markers are closely related to the prognosis and size of the tumor.

AFP can be divided into three isotypes, L1, L2 and L3, depending on the sugar chain structure and the binding affinity for one of the lectins, Lens culinaris agglutinin (LCA). AFP-L3 is also called fucosylated AFP or lectin-reactive AFP, and is a substance used for screening for hepatocellular carcinoma. AFP-L1 is mainly expressed in benign liver disease and has a non-fucosylated sugar. AFP-L2 is mainly expressed in pregnant women. AFP-L2 is also a core-fucosylated form of AFP, and its form is AFP-L3 .

In 2005, the FDA formally approved AFP-L3 as a biomarker for primary liver cancer. AFP-L3 has been used for early diagnosis of liver cancer, evaluation of therapeutic effect, and prognosis monitoring, and it is used to improve the accuracy of diagnosis compared to conventional diagnosis using AFP.

Fucose is a methylated hexose sugar and is present in several tissues and serogroups of glycoproteins called protein-bound fucose. The percentage of the AFP carbohydrate chain which has a fucose residue and which is referred to as fucose AFP (FucAFP) in the total amount of AFP is called a fucosylation index (Fuol). The fucose index has important theoretical significance and clinical application significance and may be an important index for the diagnosis and prognosis of liver cancer.

To improve the low diagnostic sensitivity of AFP, one of the markers used to diagnose liver cancer and liver disease, wako calculated AFP-L3% using AFP-L3, which is one of subtypes of AFP, as a diagnostic marker It is used as a tool to diagnose liver disease.

Specifically, the test method developed by Wako is to measure the fractional fraction (AFP-L3%) of the total AFP by the lectin reactivity in the serum. According to the Wako technique, AFP-L3% levels above 10% in patients with cirrhosis or chronic hepatitis B virus infection indicate that liver cancer is very likely, and if AFP-L3% is positive in liver cancer, The size of the lesion is relatively large and the portal vein is invaded, and it is associated with metastasis to other organs, which may be an important tool for estimating the prognosis.

Wako's device (u-TASWAKO i30) is a method for quantifying the fluorescence signal using the property of the glycoprotein called lectin, not the antibody-based AFP-L3 protein assay, The AFP-L3 type, which reacts with lectin by electrophoresis on an agarose gel containing a lectin protein, is first separated from the blood by using an antibody, The fluorescence value is converted into% and AFP-L3% is measured. However, when AFP-L3% is measured by this principle, the accuracy of quantifying AFP-L3% is decreased due to the non-specificity of the sugar affinity of the lectin protein. In addition, since Wako's separate equipment is needed, it is impossible to apply and compare the simultaneous diagnosis algorithm using multiple makers with core-lab equipment used in existing hospitals. It was not used much.

Therefore, the present inventors need an antibody capable of specifically recognizing AFP-L3 in serum and AFP-L3 detection and quantification method using the same. The development of an antibody that specifically binds to AFP-L3, a useful marker for disease diagnosis, as well as the presence or absence of core-fucosylation of AFP is necessary for the utilization and dissemination of diagnostic methods to improve the accuracy of diagnosis of liver cancer .

The present invention is based on the finding that AFP, which not only specifically binds to core fucosylated alpha-fetoprotein (AFP), such as AFP-L3, which is a useful marker of liver cancer diagnosis, -1 &lt; / RTI &gt; to selectively bind to AFP-L3 alone.

Another embodiment of the present invention is to provide an antibody that specifically recognizes only AFP-L3, distinct from AFP-L1.

Another embodiment of the present invention relates to a method for providing early detection, prognosis, and monitoring of liver cancer by selectively recognizing AFP-L3 to provide information necessary for diagnosis of disease or diagnosis of disease using an antigen-antibody reaction using the antibody .

The present invention relates to an antibody which specifically binds to AFP-L3 but which is capable of selectively binding to AFP-L3 differently from AFP which is not core-fucosylated, AFP-L3 detection or quantification method using said antibody, and And a method for providing information necessary for diagnosing or diagnosing a disease such as liver cancer using the antibody.

As used herein, the term "heavy chain" refers to a variable region domain VH comprising an amino acid sequence having a sufficient variable region sequence sufficient to confer antigen specificity, and three constant region domains CHl, CH2 and CH3 and a hinge ), And fragments thereof. The term " full-length heavy chain " In addition, the term "light chain" includes both a light chain light chain comprising a variable region domain VL comprising an amino acid sequence having a sufficient variable region sequence for imparting specificity to an antigen and a constant region domain CL and fragments thereof It is interpreted as meaning.

As used herein, the term "CDR (complementarity determining region)" refers to the amino acid sequence of the heavy chain of the immunoglobulin and the hypervariable region of the light chain. The heavy and light chains may each contain three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs may provide the major contact residues for binding of the antibody to the antigen or epitope.

In the present specification, the terms "specifically binding" or "specifically recognizing" are the same as those conventionally known to those skilled in the art, and the antigen and the antibody specifically interact to effect an immunological reaction .

One example of the present invention relates to an antibody that specifically recognizes AFP-L3. In one embodiment of the invention, the antibody is an antibody that selectively binds AFP-L3 with a higher affinity than AFP-L1, which is a non-fucosylated AFP. The anti-AFP-L3 antibody according to the present invention shows low affinity and selectivity for AFP without fucose. The structure of the core-fucosylated AFP recognized by the antibody according to the present invention is shown in Fig. Brief Description of the Drawings Fig. 1 is a schematic diagram showing the sugar structure of AFP and the developed antibody recognition performance. The high specificity and selectivity of antibodies to AFP-L3 relative to AFP-L1 suggests that the structure of the AFP protein is not currently clear, and that the two proteins have the same base sequence, It was difficult to develop an antibody specifically recognizing AFP-L3 protein having only fucose in AFP-L1 because of its difference.

Compared to lectins, especially LCA proteins, used in the AFP-L3 assay using the u-TASWAKO i30 instrument from Wako, the antibodies according to the present invention specifically recognize core-fucosylated AFP-L3 and bind it with lectin AFP-L3 can be accurately quantified compared to existing AFP-L3 diagnostic equipment, and diagnosis and prognosis of liver cancer based on this can be made.

The AFP-L3 antibody according to the present invention can be applied directly to devices for the presence or absence of a protein for the diagnosis of cancer and a method for quantitative determination of the protein, so that it is not only quick and simple, but also a multi- The AFP-L3 antibody level can be applied to the algorithm, which can provide diagnostic criteria to further improve the accuracy of early diagnosis of liver cancer.

In one example of the invention, an anti-AFP-L3 antibody or antigen-binding fragment thereof is provided. The antibody against the core-fucosylated AFP, such as AFP-L3, may comprise from 2 to 20, from 2 to 15, from 2 to 20, 10, or 2 to 5 amino acids, as an epitope (antigenic determinant site), and / or specifically binds.

Thus, the anti-AFP-L3 antibody or antigen-binding fragment thereof of the present invention may comprise an antibody or antigen binding that recognizes and / or specifically binds to the epitope described above, and an antibody or antigen binding fragment thereof competitively binding to AFP- May be at least one selected from the group consisting of

In one example of the present invention, in performing an assay on AFP-L3 based on a sandwich ELISA assay, the antibody is used as a capture antibody and natural AFP-L1 and When AFP-L1 and AFP-L3 were detected as antibodies to all AFP-L3 proteins, AFP-L1 was detected at a concentration of 50 ng / ml or more, It is preferable that the ratio of the OD value of -L3 is 1.5 times or more, for example, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, or 2.0 times or more.

In addition, the antibody according to the present invention is more preferably capable of discriminating AFP-L3 against AFP-L1 at a concentration of AFP-L3 of 50 ng / ml, 70 ng / ml, 100 ng / ml, Do.

In one example of the present invention, the anti-AFP-L3 antibody or antigen-binding fragment thereof is a complementarity determining region (CDR) of a heavy chain and has an amino acid sequence of the general formula 1 (SEQ ID NO: 1) A polypeptide having the amino acid sequence of the general formula 2 (CDR-H2), a polypeptide having the amino acid sequence of the general formula 3 (SEQ ID NO: 3) (CDR-H3) , And the like.

[Formula 1]

GX ₁ X ₂ X ₃ X ₄ X ₅ X ₆ GX ₇ G

Wherein X ₁ is F, S, Y, I, or L, X ₂ is S, P, F, or Y, X ₃ is F, S, I, or L, X ₄ is S, T , and G, or R a, X ₅ is D, N, G, or V and, X ₆ is H, or R, X ₇ is L, M, V, or I,

[Formula 2]

SX ₈ TX ₉ GGX ₁₀ X ₁₁ TWX ₁₂ X ₁₃ X ₁₄ X ₁₅ X ₁₆ X ₁₇ X ₁₈ X ₁₉ X ₂₀ X ₂₁ X ₂₂

Wherein X8 is I or T, X9 is S or N, X10 is S, G or R, X11 is G or I, X12 is Y or N, X13 is A, S or T, X14 is P or S, X15 is A or T, X16 is V or M, X17 is K, R or E, X18 is G or V, X19 is R or P, X20 is A or T X21 is T or S, X22 is I or F,

[Formula 3]

SAX ₂₃ GGWIX ₂₄ X ₂₅ DX ₂₆ X ₂₇ X ₂₈

Wherein X ₂₃ is Y or V, X ₂₄ is A or V, X ₂₅ is D, G, S or E, X ₂₆ is I, V or T, X ₂₇ is D, V or N , And X ₂₈ is A, V, S, or T.

More specifically, the anti-AFP-L3 antibody or antigen-binding fragment thereof is a complementarity determining region (CDR) of a heavy chain,

A polypeptide (CDR-H1) having an amino acid sequence selected from the group consisting of SEQ ID NOS: 7 to 11,

A polypeptide (CDR-H2) having an amino acid sequence selected from the group consisting of SEQ ID NO: 12 to SEQ ID NO: 14, and

A polypeptide (CDR-H3) having an amino acid sequence selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO: 18,

, And the like.

The anti-AFP-L3 antibody or antigen-binding fragment thereof is a complementary crystal region of a light chain, and comprises a polypeptide (CDR-L1) having the amino acid sequence of the general formula 4 (SEQ ID NO: 4), a polypeptide represented by the general formula 5 (CDR-L2) having an amino acid sequence of SEQ ID NO: 6 (CDR-L3), and a polypeptide having an amino acid sequence of the general formula 6 (SEQ ID NO: 6) (CDR-L3).

[Formula 4]

SGGX ₂₉ X ₃₀ X ₃₁ G

Wherein X29 is S or R, X30 is G, D, or S, X31 is Y, C, or H,

[Formula 5]

YX ₃₂ X ₃₃ X ₃₄ X ₃₅ RX ₃₆ X ₃₇

Wherein X32 is E or D, X33 is S, C or N, X34 is N, S, Y, D, T or H, X35 is K, R or H, X36 is P or L, X37 is S, T, P or L,

[Formula 6]

GGX ₃₈ X ₃₉ SX ₄₀ X ₄₁ X ₄₂ PGX ₄₃ FGA

Wherein X38 is S or N, X42 is I, T, or V, X43 is A or G, X40 is S, N, G, Y or V, Or T.

More specifically, the anti-AFP-L3 antibody or antigen-binding fragment thereof is a complementary crystal region of a light chain,

A polypeptide (CDR-L1) having an amino acid sequence selected from the group consisting of SEQ ID NO: 19 to SEQ ID NO: 21,

A polypeptide (CDR-L2) having an amino acid sequence selected from the group consisting of SEQ ID NO: 22 to SEQ ID NO: 27, and

A polypeptide (CDR-L3) having an amino acid sequence selected from the group consisting of SEQ ID NOS: 28 to 32,

: &Lt; / RTI &gt; &lt; RTI ID = 0.0 &gt;

In an embodiment, the anti-AFP-L3 antibody or antigen-binding fragment thereof may comprise a heavy chain complementarity determining region, a light chain complementarity determining region, or a combination thereof.

Specifically, the anti-AFP-L3 antibody or antigen-binding fragment thereof

(CDR-H1) having the amino acid sequence of SEQ ID NO: 1, a polypeptide having the amino acid sequence of SEQ ID NO: 2 (CDR-H2), and a polypeptide having the amino acid sequence of SEQ ID NO: 3 A heavy chain variable region comprising at least one heavy chain complementarity determining region selected from the group consisting of the heavy chain complementarity determining region or the at least one heavy chain complementarity determining region; And

(CDR-L3) having the amino acid sequence of SEQ ID NO: 4, a polypeptide having the amino acid sequence of SEQ ID NO: 5 (CDR-L2) and the polypeptide having the amino acid sequence of SEQ ID NO: 6 A light chain variable region comprising at least one light chain complementarity determining region selected from the group consisting of the light chain complementarity determining region or the light chain complementarity determining region;

A combination of the heavy chain complementarity determining region and the light chain complementarity determining region; or

The combination of the heavy chain variable region and the light chain variable region

. &Lt; / RTI &gt;

More specifically, the anti-AFP-L3 antibody or antigen-binding fragment thereof

A polypeptide (CDR-H1) having an amino acid sequence selected from the group consisting of SEQ ID NOS: 7 to 27, a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOS: 28 to 48, A heavy chain variable region comprising at least one heavy chain complementarity determining region selected from the group consisting of SEQ ID NO: 49 to SEQ ID NO: 61, or a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 61 (CDR-H3);

A polypeptide (CDR-L1) having an amino acid sequence selected from the group consisting of SEQ ID NOS: 62 to 68, a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOS: 69 to 85, A light chain variable region comprising at least one light chain complementarity determining region selected from the group consisting of SEQ ID NO: 86 to SEQ ID NO: 98, or a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 98 (CDR-L3), or at least one light chain complementarity determining region;

A combination of the heavy chain complementarity determining region and the light chain complementarity determining region; or

The combination of the heavy chain variable region and the light chain variable region

. &Lt; / RTI &gt;

Specifically, the heavy chain CDR of the anti-AFP-L3 antibody may be, for example, having the amino acid sequence shown in Table 1 below.

CDR-H1 SEQ ID NO: CDR-H2 SEQ ID NO: CDR-H3 SEQ ID NO: GFSFSDHGMG 7 SITSGGSGTWYAPAVKGRATI 28 SAYGGWIADDIDA 49 GFYFSDHGMG 8 SITSGGRGTWYAPAVKGRATI 29 SAYGGWIAEDIDA 50 GFSFSVHGMG 9 SITSGGSGTWYAPAVKVRATI 30 SVYGGWIADDIDA 51 GFSFSDRGMG 10 SITNGGSGTWYAPAVKGRATI 31 SAYGGWIADDVDA 52 GSSFSDHGMG 11 SITSGGGGTWYAPAVKGRATI 32 SAYGGWIASDIDA 53 GFSFSDHGLG 12 SITSGGSGIWYAPAVKGRATI 33 SAYGGWIADDIDS 54 GFSLGDHGMG 13 SITSGGSGTWYAPAVKGRTTI 34 SAYGGWIADDIDV 55 GISFSDHGMG 14 SITGGGSGTWYAPAVKGRATI 35 SAYGGWIADDTDA 56 GFSFGDHGMG 15 SITSGGSGTWYTPAVRGRATI 36 SAYGGWIADDIVA 57 GFFFSDHGMG 16 SITSGGSGTWYAPAVKGRATF 37 SAYGGWIADDIDT 58 GFPFSDHGMG 17 SITSGGSGTWYAPAMKGRATI 38 SAYGGWIADDINA 59 GYSFSDHGMG 18 SITSGGSGTWYSPAVKGRATI 39 SAYGGWIVDDIDA 60 GFSFSDHGIG 19 STTSGGSGTWYAPAVKGRATI 40 SAYGGWIAGDINA 61 GFSFSNHGMG 20 SITSGGGGIWYAPAVKGRATI 41 GLSFSVHGMG 21 SITSGGSGTWYAPAVKGRVTI 42 GFSFSDHGVG 22 SITSGGSGTWYAPAVEGRATI 43 GFSFSGHGMG 23 SITSGGSGTWNAPAVKGRATI 44 GFSFTDHGMG 24 SITSGGSGTWYAPAVKGRASI 45 GFSISDHGMG 25 SITSGGSGTWYAPTVKGRATI 46 GFPFRDHGMG 26 SITSGGSGTWYASTVKGRATI 47 GFSSSVHGMG 27 SITSGGSGTWYAPAVKGRAII 48

Specifically, the light chain CDR of the anti-AFP-L3 antibody may be, for example, having the amino acid sequence shown in Table 2 below.

CDR-L1 SEQ ID NO: CDR-L2 SEQ ID NO: CDR-L3 SEQ ID NO: SGGSGYG 62 YESNKRPS 69 GGWESSSNPGIFGA 86 SGGSDYG 63 YESDKRPS 70 GGRESSSNPGIFGA 87 SGGSSYG 64 YESTKRPS 71 GGWESNSNPGIFGA 88 SGGSGCG 65 YESSKRPS 72 GGWGSSSNPGIFGA 89 SGGRSYG 66 YESNMRPS 73 GGWESNSNPGTFGA 90 SGGSGHG 67 YESNRRPS 74 GGWESSSNPGVFGA 91 SGGRGYG 68 YESTKRPP 75 GGWESGSNPGIFGA 92 YENNKRPS 76 GGWESSSNPGIFGT 93 YESHKRPS 77 GGWESYSNPGIFGA 94 YESNKRPP 78 GGWESVSNPGIFGA 95 YESNKRPT 79 GGWESSSSPGIFGA 96 YESNKRLS 80 GGWESSNNPGIFGA 97 YESNKRPL 81 GGWESSSKPGIFGA 98 YDSNKRPS 82 YDSDKRPS 83 YECNKRPS 84 YESYKRPS 85

An example of an antibody according to the present invention may be an antibody having a combination of the light chain complementarity determining region and the light chain complementarity determining region shown in Table 11 below.

As used herein, the term "antigen binding fragment" refers to that fragment of the immunoglobulin whole structure, which refers to a portion of a polypeptide comprising a moiety capable of binding an antigen. But are not limited to, for example, scFv, (scFv) ₂ , Fab, Fab 'or F (ab') ₂ . Fab among the antigen-binding fragments has one antigen-binding site in a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain and a first constant region (CH1) of a heavy chain.

The anti-AFP-L3 antibody may be a monoclonal antibody or a polyclonal antibody. Monoclonal antibodies can be prepared by methods well known in the art. For example, it can be produced using a phage display technique. Alternatively, the anti-AFP-L3 antibody can be produced with a monoclonal antibody derived from a mouse. The finally selected antibodies can be used as humanized antibodies as well as human immunoglobulin antibodies except for the antigen binding portion. Methods for making humanized antibodies are well known in the art.

Another example provides a hybridoma that produces the antibody.

On the other hand, the anti-AFP-L3 antibody or antigen-binding fragment thereof specifically binds to AFP-L3, and thus provides a composition for the diagnosis of diseases related to AFP-L3. The disease associated with the AFP-L3 tumor marker may be liver cancer or liver related disease.

In another example, the method comprises treating the biological sample obtained from the patient with the anti-AFP-L3 antibody or antigen-binding fragment thereof; Confirming whether an antigen-antibody reaction has occurred; And determining that the patient has an AFP-L3 related disease when an antigen-antibody reaction is detected.

The step of confirming whether the antigen-antibody reaction is confirmed can be carried out through various methods known in the art. For example, it can be measured by a conventional enzyme reaction, fluorescence, luminescence and / or radiation detection, and specifically, immunochromatography, immunohistochemistry, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), Western blotting ), And the like, but the present invention is not limited thereto.

The subject to be diagnosed may be a mammal including a primate including a human, a monkey, etc., a rodent including a mouse, a rat, and the like.

The biological sample may be a cell, tissue, body fluid, plasma or blood obtained from a patient.

In another example, the heavy chain complementarity determining region, the light chain complementarity determining region, or a combination thereof of the aforementioned anti-AFP-L3 antibody; Or a polynucleotide molecule encoding a polypeptide comprising a heavy chain variable region, a light chain variable region, or a combination thereof.

Recently, a chemiluminescence immunoassay using the principle of measuring the binding between an antigen and an antibody using a chemiluminescence reaction has been recently used. Chemiluminescence is a phenomenon in which a chemiluminescent substance is excited from a low energy state to a high energy state and then returns to a ground state, thereby emitting light. This is different from fluorescence in that the energy that excites a molecule is not a light but a chemical reaction. The sensitivity is higher than the analytical method.

The antibody according to the present invention not only specifically binds to the core-fucosylated AFP (AFP-L3) but also can selectively bind to AFP-L3 selectively from the non-core-fucosylated AFP, It is possible to provide information necessary for diagnosis or diagnosis of diseases such as liver cancer.

FIG. 1 is a schematic diagram of the sugar structure of AFP and the sugar structure of core-fucosylated AFP according to the isotype of AFP and thus the developed antibody cognitive performance.
FIG. 2 is a view for explaining the principle and method of measurement AFP-L3 for existing AFP-L3 using Lectin (LCA).
Figure 3 shows the competitive immunoassay (WAKO i30) Schematic diagram of the i30 operation principle of WAKO.
FIG. 4 is a graph showing the results obtained by comparing the degree of fucosylation using Lectin Electrophoresis of a protein as a reference material with a WAKO standard calibrator according to Example 1-2. FIG.
Fig. 5 shows the result of the sugar analysis of the standard AFP protein according to Example 1-3, which is the result of MALDI-TOF MS analysis. Fig.
Fig. 6 shows the results of confirming the discrimination performance of the antibody according to the present invention for the standard substance, using the sandwich ELISA assay according to Example 4-2. Fig.

The present invention will be described in more detail with reference to the following examples. However, the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

Example  1: natural AFP- L1  And AFP- L3  Protein preparation

1-1: Natural AFP- L1  And AFP- L3  Protein preparation

Natural AFP-L1 was obtained from cord blood originated AFP protein and natural AFP-L3 was obtained by purification of liver cancer cell originated AFP protein. The natural AFP-L3 protein purification method was carried out as follows.

HCC (Hepatocellular carcinoma cell) a of the cell lines derived from Huh7 cells, 10% FBS (Fetal Bovine Serum, FBS) (Welgene, Korea), the example DMEM media (Welgene, Korea) for 37 ℃, 5% CO ₂ incubator Lt; / RTI &gt; cells. When the cultured cells were grown to a total of more than 2.5 × 10 ⁶ cells in a 150 π dish, the media was completely removed by aspirating the remaining DMEM media into the cells without closing them. The dish was washed with 1X PBS buffer (phosphate buffer solution) and the cells were grown in DMEM media without FBS for 72 hours at 37 ° C in a 5% CO ₂ incubator . The cultured medium was collected and centrifuged at 1000 rpm for 3 minutes to remove cell debris from the settled cells and obtain supernatant. The obtained supernatant was used as a purified AFP-L3 natural solution.

The above solution was added to 20X PBS stock (BioWorld, Korea) in a protein G bead (GE Healthcare Life Science, USA) in which AFP mouse-derived antibody (Boditechmed, Korea) was conjugated to the whole of the obtained purified natural AFP- Put 1/20 of the volume to make 1X PBS composition. This was slowly poured into the column containing the protein G bead. All of the natural AFP-L3 purified solution was passed through the column, washed with 1X PBS at a rate of 10 times or more of 1X PBS in a bead volume, and the washing step of the beads was carried out to remove proteins not attached to the beads. Later, the native AFP-L3 protein attached to the AFP antibody was eluted with a protein IgG elution buffer (Thermo, USA), and then purified with a 1 M Tris-HCl (pH 9.5) solution to obtain a solution containing the natural AFP- Lt; / RTI &gt; The collected protein was loaded with a 1X PBS solution in excess of the dialysis cassette, and a dialysis step was performed to replace the buffer in the dialysis cassette with 1X PBS buffer. After completion of the dialysis, the proteins were quantified by using BCA protein quantification kit (Thermo, USA) and stored frozen.

Example  1-2: Lectin In the immunoblotting method  Electrophoresis

The reference materials AFP-L1 and AFP-L3 obtained in Example 1-1 were high purity and the results of the lectin immunoblotting test showed that the AFP-L1 and AFP-L3 band positions of the Wako reference material and the band Were similar to each other. The results of the lectin immunoblot test show that the AAC-L1 and AFP-L3 band positions of the Wako reference material and the sugar assays of the self-assured reference material allow the Wako reference material AFP-L3 to have a large amount of AFP-L3 specific Which is the most abundant.

A specific lectin immunoblot test was performed by adding LCA (vectorlab, USA) at a concentration of 0.2 mg / ml to prepare a 1% agarose gel and loading a protein sample onto the gel. Samples to be loaded were AFP-L3 protein purified by Example 1-1 and AFP-L3 standard material sold by Wako Co., Ltd. for each concentration, and the gel was electrophoresed at 120 volts for 1 hour. The red gel was run for 1 hour at 40 mA using a TE77 ECL SemiDry Transfer Unit (Amersham Biosciences, UK) to transfer to the NC membrane. After transferring the NC membrane, the membrane was blocked for 1 hour, and reacted with O / N in the presence of a monoclonal antibody (1G7) antibody (Merck Millipore, USA) at 4 ° C for AFP and washed three times with PBST solution. Anti-mouse Fc HRP antibody (Thermo, USA) was reacted at room temperature for 1 hour. After washing 3 times with PBST solution, Substrate was added and images were obtained with ChemiDoc (Bio-Rad, USA).

FIG. 4 is a graph showing the results of checking the degree of fucosylation using Lectin Electrophoresis of the control protein, compared with the standard substance of WAKO, according to Example 1-2. FIG.

Example  1-3: Glycoprotein analysis of purified AFP protein

The N-glycosidase (PNGase F) enzyme was reacted with the phosphorylated buffer to separate the sugar chain from the purified natural AFP-L1, natural AFP-L3 protein purified in Example 1-1. The GP solution was reacted at room temperature for 4 hours to label the cleaved sugar chain with Girarad's reagent P (GP). GP-labeled sugar chains are mixed with dihydroxybenzoic acid (DHB), and GP-labeled sugar chains are quantified using MALDI-TOF MS. MALDI spectra were obtained using Bruker Daltonics Microflex LRF MALDI-TOF MS (Bruker, Germany). The detailed procedure was the same as that of the reference (Biotechnology Letters, October 2015, Volume 37, Issue 10, pp 2019-2025).

The results of the above experiment are shown in FIG. 5, and it is confirmed through the sugar analysis of MALDI-TOF MS analysis-standard material that AFP-L3 has a larger amount of AFP-L3 specific sugar than the purified AFP-L1 Respectively.

Example  2: Natural AFP- L3  Antibody Candidate Selection and Performance Verification

Example  2-1: Preparation of antibodies

For the preparation of antibodies, the antibody that distinguishes the AFP-L3 protein purified from the purified AFP-L3 protein for use as a control substance in the present invention in Example 1-1 was screened using phage display technology The library was constructed.

The prepared antibody library was constructed in the form of expressing antibody of ScFv form and has a complexity of ¹ × ^{10 10} . Antibody pools were screened by panning method to selectively detect AFP-L3, a control substance, and antibodies not recognizing AFP-L1 in the entire library. Panning was performed by conjugating AFP-L3 protein to magnetic beads. In order to select antibodies to AFP-L3 protein, a large amount of AFP-L1 control protein was added to the beads to bind the AFP-L3 protein A pool of well-matched antibodies was screened. Each phage clone expressing the antibody in the selected antibody pool was expressed in the form of phage displayed antibody. Screening of the phage clone was performed based on ELISA test.

Example  2-2: Immunization and cDNA library production

To select antibodies specifically binding to AFP-L3 (Ag), an animal immunocyte library was constructed. The library was obtained by obtaining mRNA from immune cells immunizing an animal with an antigen, amplifying the antibody gene through PCR using a primer combination of the antibody gene, and cloning into a vector for phage display.

Specifically, core-fucosylated AFP (AFP-L3) (Ag) was mixed with complete Freund's adjuvant and incomplete Freund's adjuvant (Sigma, USA) 5 times. The serum of the immunized animals was diluted to a concentration of 1: 100, 1: 500, 1: 2500 and 1: 12500 using PBSB (phosphate buffer containing 3% fetal bovine serum albumin) After storage, binding was confirmed by enzyme immunoassay.

In the specific enzyme immunoassay, Ag 1 ug / ml was coated overnight at 4 캜 on an ELISA plate, and diluted serum was added thereto, followed by reaction for 2 hours. After washing three times with PBST (PBS containing 0.1% tween-20), anti-chicken immunoglobulin-HRP (horse radish peroxidase) (1: 3000) was added and reacted for 1 hour. After washing three times with PBST, ABTS (Thermo, USA) was added and developed for 20 minutes. Absorbance at 405 nm was measured with a microplate reader. Preimmune serum was not bound to Ag, and animals producing sera that bind strongly to Ag were selected.

Five days after the last injection, selected chicken marrow, spleen, and Fabry cysts tissues were harvested. The collected tissues were mixed with 10 ml of TRI reagent (Molecular research center, USA), and homogenized with a homogenizer. Then, 20 ml of TRI reagent was added and centrifuged to obtain supernatant. 3 ml of 1-bromo-3-chloropropane (BCP) was added thereto, and the mixture was centrifuged to obtain a supernatant. And total RNA was precipitated with 15 ml of isopropanol. (5 min at 65 ° C; 5 min at 4 ° C; 50 min at 50 ° C; 5 min at 85 ° C) using a superscript transcription system (Invitrogen, USA) using a random hexamer as primer ; And 4 &lt; 0 &gt; C). 5 μl of the reaction solution containing the resultant cDNA of the reverse transcription reaction was loaded on 1% agarose gel and electrophoresis was performed to identify cDNA bands of various lengths. The random hexamer primer is represented by the general formula [d (N) ₆ ], and N includes all of four nucleotide sequences A (adenine), T (thiamine), G (guanine) and C .

Example  2-3: Preparation of antibody library

(1) Immuno-antibody gene amplification

The variable regions of heavy and light chains of chicken antibodies, V _H and V _L To amplify the domain, PCR reaction was performed as follows.

The PCR reaction was carried out using the primer combination shown in Table 3, which was designed for the heavy chain variable region, the light chain variable region and the scFv (single chain Fv) region linked thereto, using the cDNA prepared in Example 2-1 as a template. V _H and V _L cDNA libraries 0.5 microliters, 30 pmole forward primer, 30 pmole reverse primer, 10x PCR buffer, 200 uM dNTPs, and 0.5 μl Taq DNA polymerase were mixed and the final 50 μl was mixed, For 5 minutes, followed by 30 cycles of reaction at 94 ° C for 15 seconds, at 56 ° C for 30 seconds, and at 72 ° C for 90 seconds.

division primer Sequences (5 '-> 3') SEQ ID NO: V _H Forward &Lt; RTI ID = 99 Reverse CTGGCCGGCCTGGCCACTAGTGGAGGAGACGATGACTTCGGTCC 100 V _L Forward GTGGCCCAGGCGGCCCTGACTCAGCCGTCCTCGGTGTC 101 Reverse GGAAGATCTAGAGGACTGACCTAGGACGGTCAGG 102 scFV Forward GAGGAGGAGGAGGAGGAGGTGGCCCAGGCGGCCCTGACTCAG 103 Reverse GAGGAGGAGGAGGAGGAGGAGCTGGCCGGCCTGGCCACTAGTGGAGG 104

The PCR amplified antibody DNA was electrophoresed on 1% agarose gel, and each amplified DNA was separated according to the size and purified using a gel extraction kit (Qiagen, USA).

To obtain scFv DNA, 30 pmole forward primer, 30 pmole reverse primer, 10x PCR buffer, 200 uM dNTPs, and 0.5 uL Taq DNA polymerase were mixed with 50 ng of purified V _H , V _L 50 ng DNA as a template, Followed by reaction at 94 ° C for 5 minutes, and reaction at 94 ° C for 30 seconds, 56 ° C for 30 seconds, and 72 ° C for 2 minutes. The DNA amplified by the PCR was electrophoresed on 1% agarose gel, and the amplified DNAs were separated according to their sizes and purified using a gel extraction kit (Qiagen, USA).

(2) restriction enzyme cleavage of antibody DNA

The scFv prepared above and pComb3X (the Scripps Research Institute, CA, USA), a phagemid vector, were digested with restriction enzyme SfiI (Roche, USA).

10 ug of the PCR fragment encoding the scFv, 360 units of SfiI (Roche, USA), 20 ul of 10x buffer, and reacted overnight at 50 캜 with a final volume of 200 ul.

In addition, 20 ug of the pComb3X vector, 120 units of SfiI and 20 μl of 10 × buffer were added and reacted at 50 ° C. overnight to a final volume of 200 μl. Each of the sections cut with the restriction enzyme was electrophoresed on an agarose gel and then purified using a gel extraction kit (Qiagen, USA).

(3) Antibody DNA Ligation  &Lt; / RTI &gt;

In order to insert the scFv fragment into the pComb3X vector, 700 ng of the PCR fragment encoding the scFv cleaved with restriction enzyme SfiI and 1.4 p of pComb3X were mixed and added with T4 DNA ligase (Invitrogen, USA) Lt; / RTI &gt; Purifying the ligation mixture by ethanol precipitation method, E. coli ER2738 (New England Biolab, USA), electroporation (electroporation) into transformed by culturing under 46ug / ml carbenicillin and 70ug / ml kanamycin to 1X10 ¹⁰ complexity in Was constructed and used.

Example  2-4: anti- Ag scFV Screening of phage clones

Antibodies binding to Ag from libraries with heavy and light chains randomized in the form of scFv obtained were selected using Ag supported on solid.

Antibody binding To select, first 10 micrograms of Ag were conjugated to the magnetic beads, respectively. Antibody library DNA made to display the resultant fused scFv-type antibody with the phage coat protein PIII was transformed into E. coli ER2738 (New England Biolab) by electroporation, incubated at 37 DEG C, and then transformed into VCSM13 helper phage , USA), and then 46 ug / ml carbenicillin and 70 ug / ml kanamycin were added and cultured overnight in SB medium (30 g / L tryptone, 20 g / L yeast extract and 10 g / L MOPS, pH 7.0).

The culture solution containing the Escherichia coli and the phage obtained above was centrifuged to remove the precipitated E. coli. After recovering only the supernatant, 40 mg / ml polyethylene glycol 8000 and 30 mg / ml NaCl were added and centrifuged to collect the phage precipitated by PEG and resuspended in PBS.

Ag-phage affinity Ag was bound to the magnetic beads by reacting the Ag and the phage at room temperature for 2 hours, then washed with PBS containing 0.5% Tween 20, and immersed in 0.1 M glycine (pH 2.2) solution Eluted and neutralized with 2M Tris solution. The eluted phages were infected with E. coli ER2738 for subsequent round panning and cultured overnight to proliferate. This process was repeated four times and panning was performed. As the number of panning increases, the number of washing increases and the phages with high binding force accumulate.

Individual clones selected from the plates of the fourth pan result were incubated overnight at 37 ° C with 100 ug / ml carbenicillin, 70 ug / ml kanamycin and VCSM13 helper phage (1: 1000) in a 96 well deep well plate, The proliferation of the expressed phage was induced.

The medium obtained above was centrifuged to obtain a supernatant containing the phage. The phage prepared in the EL-coated plate coated with Ag was incubated at 37 ° C for 2 hours. The anti-M13 antibody conjugated with HRP was incubated with secondary Antibodies used as antibodies were identified by ELISA.

Example  2-5: Sequence analysis of selected antibodies

In Example 2-4, ER2738 having a clone showing a positive reaction under selection conditions was cultured overnight in SB medium, followed by centrifugation to obtain E. coli. Plasmid DNA was obtained using a DNA mini prep kit (Jinol, Korea) and the nucleotide sequences thereof were analyzed. Sequencing primers were sequenced using sequencing primer sequence numbers (forward) and (reverse) primers as shown in Table 4 below.

(SEQ ID NO: 105) ACACTTTATGCTTCCGGCTC Reverse (SEQ ID NO: 106) CAAAATCACCGGAACCAGAG

Example  2-6: ELISA test for antibody selection

A monoclonal HA antibody (Abcam, UK) was coated on the ELISA plate to recognize HA tag, and each well was reacted with BSA solution for 1 hour at 37 ° C. Each clone of the pool selected by the preceding panning operation was further reacted with E. coli culture media expressed by phage displayed scFv for 2 hours at 37 ° C. After washing with 3 PBST solutions, the purified Control AFP-L3 and Control AFP-L1 protein (10 ug / ml) were reacted at 37 ° C for 2 hours on an antibody-coated plate. After washing with PBST solution three times, the antibodies that had been biotinylated with AFP-recognizing antibody were reacted at 37 ° C for 2 hours. After washing with 3 PBST solutions, avidin-HRP antibody was reacted at 37 ° C for 1 hour. After washing again with 3 PBST solutions, the cells are developed with ABTS solution, reacted at room temperature for 30 minutes, and then the OD value of each well is measured at OD = 450 nM. Milipore, ST1673, and Anti-AFP Mouse mAb (1G7) were used as the detection antibody, which did not discriminate between AFP-L1 and AFP-L3.

 The candidate E. coli-expressing antibodies used in the above test were 5 species, and the antibodies were named L3-1 A4, L3P2-D5, 1-1 H1, L3-2 C10, 2-1 B8 L3-2 C10, 2-1 B8) were selected. It is the average value of the experiment of 3 repetitions. During the screening, the candidate antibody was applied as capture antibody.

division BSA AFP-L1 AFP-L3 L3-1 A4 0.14 0.13 0.13 L3P2-D5 0.15 0.23 0.31 1-1 H1 0.14 0.23 0.25 L3-2 C10 0.13 1.16 3.46 2-1 B8 0.16 0.18 0.63

E. coli expressed antibodies were screened by sandwich ELISA to select the final two candidate antibodies (L3-2 C10, 2-1 B8) that recognize the reference AFP-L3 as affinity.

Example  3: Sandwich ELISA  AFP- L3  Identifying and Selecting Distinctive Performance

To evaluate the binding affinity of the AFP-L3 antibody to the presence or absence of the sugar using the sandwich ELISA.

Similar to Example 2, the antibody was screened by coating a monoclonal HA antibody on the plate, and the BSA solution was reacted at 37 ° C for 1 hour to perform blocking. The phage displayed scFv (C10, B8) antibody was then reacted in the wells at 37 ° C for 2 hours. AFP-L1 and AFP-L3, in the form of protein-free sugars, were treated with N-glycosidase PNGase enzyme (NEB, USA) with control AFP-L1 and control AFP- The reaction was carried out in the same manner as in Example 2-6, and the average values of the triplicate experiments were shown.

It was confirmed that the selective antibody binds weakly to the standard substance from which the sugar of AFP-L3 was removed than the protein which did not remove the sugar. It was confirmed that the screening antibody recognizes the sugar (including the epitope AFP-L3 part) and binds affinity to the AFP-L3 reference material. The antibody IgG O.D values are shown in Table 6 below.

antigen L3-2 C10 antibody L2-1 B8 antibody AFP-L1 0.61 0.23 AFP-L1 (sugar removal) 0.30 0.18 AFP-L3 2.08 1.23 AFP-L3 (sugar removal) 1.66 0.61

The screening antibodies C10 and B8 were found to weakly bind to AFP-L3-depleted standards than to proteins that did not remove sugars. This confirms that the selective antibody recognizes the sugar (including the epitope AFP-L3 part) and binds affinity to the AFP-L3 reference material.

Example  4: Natural AFP- L3  Selection of antibody candidates and performance confirmation (application of antibody expressing purified animal cells IgG ))

Example  4-1: Application of expressing antibody using purified animal cells

Immunoglobulin (IgG) protein was produced for the determination of the affinity of the antibody obtained in Example 2 and the activity analysis. Specifically, fragments of the variable region and the constant region of the heavy chain and light chain from pComb3x containing scFv were obtained through PCR under the same conditions as in Example 2 using the primer combination shown in Table 7 below.

That is, each variable region and constant region (CH and Ck) were subjected to PCR using HC and LC primer combinations shown in Table 7 below to obtain heavy and light chains, respectively. The pcDNAATM3.3-TOPO.RTM. Cloning kit and pOptiTMVEC- And transferred to mammalian cell expression plasmids using a kit (Invitrogen, USA). 1 μl of each vector (pcDNATM3.3-TOPO® vector and pOptiTM VEC-TOPO® vector) and a slice were added to a buffer solution containing 200 mM NaCl and 10 mM MgCl ₂ in a total volume of 6 μl, and reacted at room temperature for 5 minutes. DH 5a Escherichia coli competent cells were transformed with heat shock to obtain colonies, followed by mass culture to obtain plasmids.

division primer Sequences (5 '-> 3') SEQ ID NO: V _H Forward GCTAGCCGCCACCATGGGC 107 Reverse AGGGGCCCTTGGTGGAGGCCTGGCCGGCCTGGCCACT 108 C _H Forward GCCTCCACCAAGGGCCCCTC 109 Reverse CGGGATCCCTTGCCGGCCGT 110 HC Forward GCTAGCCGCCACCATGGGC 111 Reverse CGGGATCCCTTGCCGGCCGT 112 V _L Forward AAGCTTGCCGCCACCATG 113 Reverse AGGGGGCGGCCACGGTCCGGGAAGATCTAGAGGACTG 114 C _k Forward CGGACCGTGGCCGCCCCCTC 115 Reverse GCTCTAGACTAGCACTCGC 116 LC Forward AAGCTTGCCGCCACCATG 117 Reverse GCTCTAGACTAGCACTCGC 118

The plasmid was used to transfect HEK293F cells (Invitrogen, USA), and incubated for 7 days. The obtained antibody was purified using protein A column (GE, USA). The culture was loaded on the column, and the antibody (IgG) in the culture broth was bound to Protein A and eluted with 20 mM sodium citrate buffer (pH 3.0). As shown in FIG. 6, molecular weight and high purity were confirmed by SDS-PAGE of light and heavy chains in accordance with the theoretical calculated values of light chain (25 kDa) and heavy chain (50 kDa).

Example  4-2: Identification of Antibody Separation Performance against Control Reference Substance

The selective sandwich ELISA method was performed by expressing a candidate antibody selectively recognizing AFP-L3 protein in an IgG form and then coating it on an ELISA plate at a concentration of 5 ug / ml. Each well was blotted with BSA solution, and the Control AFP-L3 and Control AFP-L1 protein (10 ug / ml) purified in Example 1 were reacted at 37 ° C for 2 hours on an antibody-coated plate. After washing three times with PBST solution, the detection antibody was reacted at 37 ° C for 2 hours, and then re-washed with PBST solution three times. Then, secondary antibody labeled with HRP enzyme in avidin reacted with Biotin was incubated at 37 ° C And reacted for 1 hour. After washing with 3 PBST solutions, the cells are developed with ABTS solution, reacted at room temperature for 30 minutes, and OD value of each well is measured at OD = 450 nM. 3 is the mean value of the repeated experiments. The test results are shown in Table 8 and FIG. Milipore, ST1673, and Anti-AFP Mouse mAb (1G7) were used as the detection antibody, which did not discriminate between AFP-L1 and AFP-L3.

antigen L3-2 C10 antibody L2-1 B8 antibody AFP-L1 50 ng / ml 0.23 No data AFP-L1 100 ng / ml 0.16 0.41 AFP-L1 1000 ng / ml 0.63 0.13 AFP-L3 50 ng / ml 0.37 No data AFP-L3 100 ng / ml 0.34 0.33 AFP-L3 1000 ng / ml 1.97 0.89

As shown in the results of Table 8 and FIG. 6, it was confirmed that the L3-2C10 antibody, one of the IgG type selection antibodies, had a higher affinity for AFP-L3 than the AFP-L3, and L3- 2 &lt; / RTI &gt; C10 antibody showed a discriminative performance at a relatively low concentration of 50 ng / ml.

Example  4-3: Identification of antibodies to Wako calibrator

An antibody selectively recognizing AFP-L3 obtained in Example 2 was expressed in the form of IgG, purified, and then coated on an ELISA plate at a concentration of 5 ug / ml. Each well was blotted with BSA solution, and Wako calibrator AFP-L3 (100 ng / ml) and Wako Calibrator AFP-L1 protein (100 ng / ml) were reacted at 37 ° C for 2 hours on an antibody-coated plate. After washing with 3 PBST solutions, the detection antibody was reacted at 37 ° C for 2 hours, then washed with 3 PBST solutions, and then the secondary antibody labeled with HRP enzyme in avidin reacted with Biotin was incubated at 37 ° C for 1 hour Lt; / RTI &gt; After washing with 3 PBST solutions, the cells are developed with ABTS solution, reacted at room temperature for 30 minutes, and OD value of each well is measured at OD = 450 nM. It is the average value of 3 repeated experiments. Milipore, ST1673, and Anti-AFP Mouse mAb (1G7) were used as the detection antibody, which did not discriminate between AFP-L1 and AFP-L3.

antigen L3-2 C10 antibody Wako AFP-L1 (100 ng / ml) 0.21 Wako AFP-L3 (100 ng / ml) 0.32

In the case of the screening antibody L3-2 C10, it was confirmed that AFP-L1 and AFP-L3, which were sold by Wako, were distinguished from AFP-L1 and AFP-L3 at a concentration range of 100 ng / ml.

That is, by performing immunoassay using the screening antibody L3-2 C10 according to the present invention, the target detection antigens were divided into two groups, and as a result, the control AFP-L1 and AFP- L3 protein, and AFP-L1 and AFP-L3 proteins, which are sales standard products of Wako Co., respectively. As a result, AFP-L1 and AFP-L3 were distinguished from each other.

Example  5: Comparison with Wako system using ELISA

5-1: Preparation of sample

As a sandwich ELISA assay, control AFP-L1 and AFP-L3 proteins purified and purified in Example 1 were mixed so that the total protein concentration was 200 ng / ml. At this time, the basic buffer conditions of the mixture were AFP depleted serum was used to prepare mixed AFP protein solution.

5-2: ELSIA Analysis by method

The control AFP-L1 and AFP-L3 proteins purified and obtained in Example 1 were analyzed and analyzed by an ELISA analysis method using an antibody according to the present invention.

Specifically, the ELISA analysis was carried out on the sample prepared in 5-1 above according to the method of Example 2-6, and the results of the analysis are shown in Table 10 below.

5-3: Analysis by WAKO analysis machine

The control AFP-L1 and AFP-L3 proteins purified and purified in Example 1 were analyzed by ELISA analysis using a uTas i30 instrument from Wako.

The value of AFP-L3 (%) was obtained by measuring with a Wako uTas i30 instrument, and the IgG of the L3-2 C10 antibody obtained in the present invention was coated, and each sample was identified as AFP through a sandwich ELISA Sandwich ELISA test was performed by applying an antibody pair for recognition of core-fucosylated AFP (AFP-L3) using antibody pair and L3-2 C10.

The amount of total AFP protein calculated by applying the standard curve drawn at this time and the amount of core-fucosylated AFP measured using L3-2 C10 were determined, and the result was expressed as% Pearson correlation coefficients were calculated to confirm the relationship.

division Total AFP protein concentration (ng / mL) AFP-L1 protein (ng / mL) AFP-L3 protein (ng / mL) AFP-L3 (%) - wako uTas measurement value AFP-L3 (%) - developed antibody ELISA measurement value Sample 1 200 20 180 33.6 11,6 Sample 2 200 100 100 55.4 16.2 Sample 3 200 180 20 81.4 33.2

The correlation score between the uTas measurement value and the developed antibody measurement value was 0.96. From the measured values of AFP-L3% measured for the control AFP-L1 and AFP-L3 proteins purified in Example 1, which is the same assay sample, It can be confirmed that there is a high correlation between the immunoassay using the developed antibody according to the present invention and the assay using the uTas instrument of WAKO.

Example  6: Various antibody production and activity confirmation

6-1: Antibody production

A screening antibody library was prepared by random mutagenesis of the screening antibody for screening of antibodies and a candidate antibody was screened.

The scFv sequence of the selected C10 antibody was used as a template and the antibody gene was subjected to PCR using the primers shown in SEQ ID NOS: 103 and 104. In order to introduce random mutagenesis into the base sequence of the antibody upon amplification of the antibody, random mutagenesis was introduced into the antibody gene using the random mutagenesis kit (Jenabioscience, Germany) as described in the kit. The antibody gene amplified by the above kit was subjected to restriction enzyme digestion in the same manner as described in Example 2-3: Preparation of Antibody Library, and a C10 random mutagenized antibody library was prepared. ⁹ complexity.

Antibodies binding to Ag from libraries with heavy and light chains randomized in the form of scFv obtained were selected using Ag supported on solid.

6-2: Antibody screening

First, 10 micrograms of Ag were conjugated to the magnetic beads, respectively.

Antibody library DNA made to display the resultant fused scFv-type antibody with the phage coat protein PIII was transformed into E. coli ER2738 (New England Biolab) by electroporation, incubated at 37 DEG C, and then transformed into VCSM13 helper phage , USA), and then 46 ug / ml carbenicillin and 70 ug / ml kanamycin were added and cultured overnight in SB medium (30 g / L tryptone, 20 g / L yeast extract and 10 g / L MOPS, pH 7.0).

The culture solution containing the Escherichia coli and the phage obtained above was centrifuged to remove the precipitated E. coli. After recovering only the supernatant, 40 mg / ml polyethylene glycol 8000 and 30 mg / ml NaCl were added and centrifuged to collect the phage precipitated by PEG and resuspended in PBS.

Ag-phage affinity Ag was bound to the magnetic beads by reacting the Ag and the phage at room temperature for 2 hours, then washed with PBS containing 0.5% Tween 20, and immersed in 0.1 M glycine (pH 2.2) solution Eluted and neutralized with 2M Tris solution. The eluted phages were infected with E. coli ER2738 for subsequent round panning and cultured overnight to proliferate. This process was repeated four times and panning was performed. As the number of panning increases, the number of washing increases and the phages with high binding force accumulate.

Individual clones selected from the plates of the fourth pan result were incubated overnight at 37 ° C with 100 ug / ml carbenicillin, 70 ug / ml kanamycin and VCSM13 helper phage (1: 1000) in a 96 well deep well plate, The proliferation of the expressed phage was induced.

6-3: Activity confirmation test

Monoclonal HA antibody was coated on the plate and the BSA solution was reacted at 37 DEG C for 1 hour in order to identify and select the activity of the antibody similarly to Example 2-6. Control AFP-L1 and control AFP-L3 obtained in Example 1 were treated at a concentration of 1 ug / ml at 37 ° C for 2 hours, and washed three times with PBST solution.

Each of the obtained phage antibody cultures was treated and reacted at 37 ° C for 2 hours. Then, PBST solution was washed three times, and the antibody that had been biotinylated was reacted at 37 ° C for 2 hours. Milipore, ST1673, and Anti-AFP Mouse mAb (1G7) were used as the detection antibody, which did not discriminate between AFP-L1 and AFP-L3. After washing with 3 PBST solutions, avidin-HRP antibody was reacted at 37 ° C for 1 hour. After washing again with 3 PBST solutions, the cells were developed with ABTS solution, reacted at room temperature for 30 minutes, and the OD value of each well was measured at OD = 450 nM. Thereafter, the gene analysis of the antibody proceeded in the same manner as in Example 2-5, and antibodies having different C10 and CDR sequences were selected with similar performance to C10.

Antibodies listed in Table 11 were screened for antibodies having different ratios of CDRs with a ratio of O.D value of AFP-L3 O.D divided by AFP-L1 O.D value of 1.5 or more.

NO CDR-L1 CDR-L3 CDR-L3 CDR-H1 CDR-H2 CDR-H3 O.D. (Ratio) One SGGSGYG YESNKRPS GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 3.39 2 SGGSGYG YESNKRPS GGRESSSNPGIFGA GFYFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIAEDIDA 2.57 3 SGGSGYG YESDKRPS GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 2.35
4 SGGSGYG YESNKRPS GGWESNSNPGIFGA GFSFSDHGMG SITSGGRGTWYAPAVKGRATI SAYGGWIADDIDA 2.19 5 SGGSGYG YESTKRPS GGRESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 2.21 6 SGGSGYG YESSKRPS GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 2.04 7 SGGSGYG YESNKRPS GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKVRATI SAYGGWIADDIDA 2.89 8 SGGSGYG YESNMRPS GGWGSSSNPGIFGA GFSFSDHGMG SITNGGSGTWYAPAVKGRATI SVYGGWIADDIDA 2.68 9 SGGSGYG YESNKRPS GGWESSSNPGIFGA GFSFSVHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 2.68 10 SGGSGYG YESNRRPS GGWESNSNPGTFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDVDA 2.69 11 SGGSGYG YESNKRPS GGWESNSNPGIFGA GFSFSDRGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 2.57 12 SGGSGYG YESNKRPS GGWESSSNPGIFGA GSSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIASDIDA 2.90 13 SGGSGYG YESSKRPS GGWESSSNPGVFGA GFSFSDHGLG SITSGGGGTWYAPAVKGRATI SAYGGWIADDIDA 2.23 14 SGGSGYG YESNKRPS GGRESSSNPGIFGA GFSLGDHGMG SITSGGSGIWYAPAVKGRATI SAYGGWIADDIDA 2.89 15 SGGSGYG YESNKRPS GGWESSSNPGIFGA GISFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDS 2.89 16 SGGSGYG YESNKRPS GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDV 2.11 17 SGGSGYG YESTKRPP GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRTTI SAYGGWIADDIDA 2.22 18 SGGSGYG YESNKRPS GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDTDA 3.58 19 SGGSGYG YESNKRPS GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIVA 2.06 20 SGGSGYG YENNKRPS GGWESGSNPGIFGA GFSFGDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 2.15 21 SGGSGYG YESNKRPS GGWESSSNPGIFGA GFSFSDHGMG SITSGGGGTWYAPAVKGRATI SAYGGWIADDIDA 2.18 22 SGGSGYG YESNKRPS GGWESNSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 2.19 23 SGGSGYG YESNKRPS GGWESSSNPGIFGA GFSFSDHGMG SITGGGSGTWYAPAVKGRATI SAYGGWIADDIDS 2.16 24 SGGSGYG YESNKRPS GGWESSSNPGIFGA GFSFSDRGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 1.82 25 SGGSGYG YESNKRPS GGWESSSNPGIFGA GFFFSDHGMG SITSGGSGTWYTPAVRGRATI SAYGGWIADDIDA 3.16 26 SGGSGYG YESHKRPS GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 2.17 27 SGGSGYG YESNKRPS GGWESSSNPGIFGA GFPFSDHGMG SITSGGSGTWYAPAVKGRATF SAYGGWIADDIDA 3.07 28 SGGSGYG YESNKRPS GGRESSSNPGIFGA GYSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDVDA 2.73 29 SGGSGYG YESNKRPS GGWESSSNPGIFGA GFSFSDHGIG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 2.02 30 SGGSGYG YESNKRPP GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAMKGRATI SAYGGWIADDIDA 2.01 31 SGGSGYG YESNKRPT GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 2.30 32 SGGSGYG YESNKRPS GGWESSSNPGIFGA GFSFSNHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDT 2.39 33 SGGSGYG YENNKRPS GGWESNSNPGIFGA GFSFSVHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIVA 2.45 34 SGGSGYG YESNKRPS GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIAEDIDA 2.04 35 SGGSGYG YESNKRPS GGWESSSNPGIFGA GLSFSVHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 2.22 36 SGGSGYG YESNKRPS GGWESGSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRTTI SAYGGWIADDINA 2.29 37 SGGSGYG YESNKRPS GGWESSSNPGIFGT GFSFSDHGMG SITSGGSGTWYSPAVKGRATI SAYGGWIADDIDA 2.36 38 SGGSGYG YESSKRPS GGWESSSNPGIFGA GFSFSDHGMG STTSGGSGTWYAPAVKGRATI SAYGGWIADDIDT 2.12 39 SGGSGYG YESNKRLS GGWGSSSNPGIFGA GFSFSDHGMG SITSGGGGIWYAPAVKGRATI SAYGGWIADDIDA 2.12 40 SGGSGYG YESNKRPS GGWESSSNPGIFGA GFSFSDHGVG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 2.15 41 SGGSDYG YESNKRPL GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDINA 3.04 42 SGGSGYG YESNKRPT GGRESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 2.64 43 SGGSGYG YESNKRPS GGWESSSNPGVFGA GFSFSGHGMG SITSGGSGTWYAPAVKGRTTI SAYGGWIADDIDA 2.24 44 SGGSGYG YESNKRPS GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRVTI SAYGGWIADDIDA 2.65 45 SGGSGYG YESNKRPS GGRESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 2.24 46 SGGSGYG YESNKRPS GGWESYSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRASI SAYGGWIADDIDA 1.67 47 SGGSGYG YESNKRPS GGWESYSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVEGRATI SAYGGWIADDIDA 1.70 48 SGGSGYG YESNKRPS GGWESSSNPGIFGA GFSFSDHGVG SITSGGSGTWYAPAVKGRTTI SAYGGWIADDIDA 1.66 49 SGGSGYG YDSNKRPS GGRESSSNPGIFGA GFSFSDRGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 1.81 50 SGGSGYG YESNKRPT GGRESSSNPGIFGA GFSFTDHGMG SITSGGSGTWNAPAVKGRATI SAYGGWIADDIDA 2.28 51 SGGSGYG YESNRRPS GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 2.09 52 SGGSGYG YDSDKRPS GGRESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 1.93 53 SGGSSYG YESNKRPS GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIVDDIDA 2.62 54 SGGSGYG YESNKRPS GGWESVSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 1.67 55 SGGSGCG YECNKRPS GGWESSSNPGIFGA GFSISDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIVA 2.12 56 SGGSGYG YESNKRPS GGWESNSNPGIFGA GFPFRDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDT 2.31 57 SGGSGYG YESNKRPS GGWESSSNPGVFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 1.80 58 SGGSGYG YENNKRPS GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPTVKGRATI SAYGGWIADDIDA 1.76 59 SGGSGYG YESYKRPS GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYASTVKGRATI SAYGGWIADDIDA 1.66 60 SGGSGYG YESNKRPL GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 1.71 61 SGGRSYG YESNKRPS GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 1.67 62 SGGSGYG YESNKRPS GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRAII SAYGGWIADDIDA 2.05 63 SGGSGYG YESNKRPS GGWESSSSPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 2.00 64 SGGSGYG YENNKRPS GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDVDA 2.31 65 SGGSGYG YESNKRPS GGWESSSNPGIFGA GFSSSVHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 2.00 66 SGGSGYG YESYKRPS GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 2.15 67 SGGSGHG YESNKRPS GGWESSSNPGIFGA GFSFSDHGLG SITSGGSGTWYAPAVKGRATI SAYGGWIAGDINA 2.03 68 SGGSGYG YESNKRPS GGWESSSNPGIFGA GFSLGDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 2.86 69 SGGSGYG YESNKRPS GGWESSNNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 2.09 70 SGGSGYG YESNKRPT GGWESSSKPGIFGA GFSFSDHGLG SITSGGSGTWYAPAVKGRATI SAYGGWIADDVDA 2.96 71 SGGSGYG YESNKRPP GGWESSSNPGIFGA GFSFSDRGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 2.44 72 SGGRGYG YESNKRPS GGWESSSNPGIFGA GFSFSDHGMG SITSGGSGTWYAPAVKGRATI SAYGGWIADDIDA 2.36

Table 12 shows the OD values measured by the actual ELISA analysis of five representative antibodies among the antibodies listed in Table 11, and the experiment was carried out in the same manner as in Example 2-6.

NO AFP-L1 (O.D.) AFP-L3 (O.D.) 10 1.5036 3.0411 35 1.065 2.3648 37 0.9962 2.3472 55 1.0814 2.2879 61 1.4043 2.3391

<110> SK TELECOM CO., LTD. <120> Antibody specifically binding to core fucosylated          alpha-fetoprotein and use thereof <130> DPP20161737 <160> 118 <170> Kopatentin 1.71 <210> 1 <211> 10 <212> PRT &Lt; 213 > polypeptide (CDR-H1) <220> <221> UNSURE <222> (2) <223> X is F, S, Y, I or L <220> <221> UNSURE <222> (3) <223> X is S, P, F or Y <220> <221> UNSURE <222> (4) <223> X is F, S, I or L <220> <221> UNSURE <222> (5) <223> X is S, T, G, or R <220> <221> UNSURE <222> (6) <223> X is D, N, G, or V <220> <221> UNSURE <222> (7) <223> X is H or R <220> <221> UNSURE <222> (9) <223> X is L, M, V, or I <400> 1 Gly Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Gly   1 5 10 <210> 2 <211> 21 <212> PRT &Lt; 213 > polypeptide (CDR-H2) <220> <221> UNSURE <222> (2) <223> X is I or T <220> <221> UNSURE <222> (4) <223> X is S, or N <220> <221> UNSURE <222> (7) <223> X is S, G or R <220> <221> UNSURE <222> (8) <223> X is G or I <220> <221> UNSURE <222> (11) <223> X is Y or N <220> <221> UNSURE <12> <223> X is A, S or T <220> <221> UNSURE <222> (13) <223> X is P or S <220> <221> UNSURE <222> (14) <223> X is A or T <220> <221> UNSURE &Lt; 222 > (15) <223> X is V or M <220> <221> UNSURE <222> (16) <223> X is K, R or E <220> <221> UNSURE <222> (17) <223> X is G, or V <220> <221> UNSURE <222> (18) <223> X is R or P <220> <221> UNSURE <222> (19) <223> X is A or T <220> <221> UNSURE <20> <223> X is T or S <220> <221> UNSURE <222> (21) <223> X is I or F <400> 2 Ser Xaa Thr Xaa Gly Gly Xaa Xaa Thr Trp Xaa Xaa Xaa Xaa Xaa Xaa   1 5 10 15 Xaa Xaa Xaa Xaa Xaa              20 <210> 3 <211> 13 <212> PRT &Lt; 213 > polypeptide (CDR-H3) <220> <221> UNSURE <222> (3) <223> X is Y or V <220> <221> UNSURE <222> (8) <223> X is A or V <220> <221> UNSURE <222> (9) <223> X is D, G, S or E <220> <221> UNSURE <222> (11) <223> X is I, V or T <220> <221> UNSURE <12> <223> X is D, V or N <220> <221> UNSURE <222> (13) <223> X is A, V, S or T <400> 3 Ser Ala Xaa Gly Gly Trp Ile Xaa Xaa Asp Xaa Xaa Xaa   1 5 10 <210> 4 <211> 7 <212> PRT &Lt; 213 > polypeptide (CDR-L1) <220> <221> UNSURE <222> (4) <223> X is S or R <220> <221> UNSURE <222> (5) <223> X is G, D, or S <220> <221> UNSURE <222> (6) <223> X is Y, C, or H <400> 4 Ser Gly Gly Xaa Xaa Xaa Gly   1 5 <210> 5 <211> 8 <212> PRT &Lt; 213 > polypeptide (CDR-L2) <220> <221> UNSURE <222> (2) <223> X is E or D <220> <221> UNSURE <222> (3) <223> X is S, C or N <220> <221> UNSURE <222> (4) <223> X is N, S, Y, D, T or H <220> <221> UNSURE <222> (5) <223> X is K, R or H <220> <221> UNSURE <222> (7) <223> X is P or L <220> <221> UNSURE <222> (8) <223> X is S, T, P or L <400> 5 Tyr Xaa Xaa Xaa Xaa Arg Xaa Xaa   1 5 <210> 6 <211> 14 <212> PRT &Lt; 213 > polypeptide (CDR-L3) <220> <221> UNSURE <222> (3) <223> X is W or R <220> <221> UNSURE <222> (4) <223> X is E or G <220> <221> UNSURE <222> (6) <223> X is S, N, G, Y or V <220> <221> UNSURE <222> (7) <223> X is S or N <220> <221> UNSURE <222> (8) <223> X is I, T, or V <220> <221> UNSURE <222> (11) <223> X is A or T <400> 6 Gly Gly Xaa Xaa Ser Xaa Xaa Xaa Pro Gly Xaa Phe Gly Ala   1 5 10 <210> 7 <211> 10 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H1 <400> 7 Gly Phe Ser Phe Ser Asp His Gly Met Gly   1 5 10 <210> 8 <211> 10 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H1 <400> 8 Gly Phe Tyr Phe Ser Asp His Gly Met Gly   1 5 10 <210> 9 <211> 10 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H1 <400> 9 Gly Phe Ser Phe Ser Val His Gly Met Gly   1 5 10 <210> 10 <211> 10 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H1 <400> 10 Gly Phe Ser Phe Ser Asp Arg Gly Met Gly   1 5 10 <210> 11 <211> 10 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H1 <400> 11 Gly Ser Ser Phe Ser Asp His Gly Met Gly   1 5 10 <210> 12 <211> 10 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H1 <400> 12 Gly Phe Ser Phe Ser Asp His Gly Leu Gly   1 5 10 <210> 13 <211> 10 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H1 <400> 13 Gly Phe Ser Leu Gly Asp His Gly Met Gly   1 5 10 <210> 14 <211> 10 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H1 <400> 14 Gly Ile Ser Phe Ser Asp His Gly Met Gly   1 5 10 <210> 15 <211> 10 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H1 <400> 15 Gly Phe Ser Phe Gly Asp His Gly Met Gly   1 5 10 <210> 16 <211> 10 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H1 <400> 16 Gly Phe Phe Phe Ser Asp His Gly Met Gly   1 5 10 <210> 17 <211> 10 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H1 <400> 17 Gly Phe Pro Phe Ser Asp His Gly Met Gly   1 5 10 <210> 18 <211> 10 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H1 <400> 18 Gly Tyr Ser Phe Ser Asp His Gly Met Gly   1 5 10 <210> 19 <211> 10 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H1 <400> 19 Gly Phe Ser Phe Ser Asp His Gly Ile Gly   1 5 10 <210> 20 <211> 10 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H1 <400> 20 Gly Phe Ser Phe Ser Asn His Gly Met Gly   1 5 10 <210> 21 <211> 10 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H1 <400> 21 Gly Leu Ser Phe Ser Val His Gly Met Gly   1 5 10 <210> 22 <211> 10 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H1 <400> 22 Gly Phe Ser Phe Ser Asp His Gly Val Gly   1 5 10 <210> 23 <211> 10 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H1 <400> 23 Gly Phe Ser Phe Ser Gly His Gly Met Gly   1 5 10 <210> 24 <211> 10 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H1 <400> 24 Gly Phe Ser Phe Thr Asp His Gly Met Gly   1 5 10 <210> 25 <211> 10 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H1 <400> 25 Gly Phe Ser Ile Ser Asp His Gly Met Gly   1 5 10 <210> 26 <211> 10 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H1 <400> 26 Gly Phe Pro Phe Arg Asp His Gly Met Gly   1 5 10 <210> 27 <211> 10 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H1 <400> 27 Gly Phe Ser Ser Ser Val His Gly Met Gly   1 5 10 <210> 28 <211> 21 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H2 <400> 28 Ser Ile Thr Ser Gly Gly Ser Gly Thr Trp Tyr Ala Pro Ala Val Lys   1 5 10 15 Gly Arg Ala Thr Ile              20 <210> 29 <211> 21 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H2 <400> 29 Ser Ile Thr Ser Gly Gly Arg Gly Thr Trp Tyr Ala Pro Ala Val Lys   1 5 10 15 Gly Arg Ala Thr Ile              20 <210> 30 <211> 21 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H2 <400> 30 Ser Ile Thr Ser Gly Gly Ser Gly Thr Trp Tyr Ala Pro Ala Val Lys   1 5 10 15 Val Arg Ala Thr Ile              20 <210> 31 <211> 21 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H2 <400> 31 Ser Ile Thr Asn Gly Gly Ser Gly Thr Trp Tyr Ala Pro Ala Val Lys   1 5 10 15 Gly Arg Ala Thr Ile              20 <210> 32 <211> 21 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H2 <400> 32 Ser Ile Thr Ser Gly Gly Gly Gly Thr Trp Tyr Ala Pro Ala Val Lys   1 5 10 15 Gly Arg Ala Thr Ile              20 <210> 33 <211> 21 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H2 <400> 33 Ser Ile Thr Ser Gly Gly Ser Gly Ile Trp Tyr Ala Pro Ala Val Lys   1 5 10 15 Gly Arg Ala Thr Ile              20 <210> 34 <211> 21 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H2 <400> 34 Ser Ile Thr Ser Gly Gly Ser Gly Thr Trp Tyr Ala Pro Ala Val Lys   1 5 10 15 Gly Arg Thr Thr Ile              20 <210> 35 <211> 21 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H2 <400> 35 Ser Ile Thr Gly Gly Gly Ser Gly Thr Trp Tyr Ala Pro Ala Val Lys   1 5 10 15 Gly Arg Ala Thr Ile              20 <210> 36 <211> 21 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H2 <400> 36 Ser Ile Thr Ser Gly Gly Ser Gly Thr Trp Tyr Thr Pro Ala Val Arg   1 5 10 15 Gly Arg Ala Thr Ile              20 <210> 37 <211> 21 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H2 <400> 37 Ser Ile Thr Ser Gly Gly Ser Gly Thr Trp Tyr Ala Pro Ala Val Lys   1 5 10 15 Gly Arg Ala Thr Phe              20 <210> 38 <211> 21 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H2 <400> 38 Ser Ile Thr Ser Gly Gly Ser Gly Thr Trp Tyr Ala Pro Ala Met Lys   1 5 10 15 Gly Arg Ala Thr Ile              20 <210> 39 <211> 21 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H2 <400> 39 Ser Ile Thr Ser Gly Gly Ser Gly Thr Trp Tyr Ser Pro Ala Val Lys   1 5 10 15 Gly Arg Ala Thr Ile              20 <210> 40 <211> 21 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H2 <400> 40 Ser Thr Thr Ser Gly Gly Ser Gly Thr Trp Tyr Ala Pro Ala Val Lys   1 5 10 15 Gly Arg Ala Thr Ile              20 <210> 41 <211> 21 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H2 <400> 41 Ser Ile Thr Ser Gly Gly Gly Gly Ile Trp Tyr Ala Pro Ala Val Lys   1 5 10 15 Gly Arg Ala Thr Ile              20 <210> 42 <211> 21 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H2 <400> 42 Ser Ile Thr Ser Gly Gly Ser Gly Thr Trp Tyr Ala Pro Ala Val Lys   1 5 10 15 Gly Arg Val Thr Ile              20 <210> 43 <211> 21 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H2 <400> 43 Ser Ile Thr Ser Gly Gly Ser Gly Thr Trp Tyr Ala Pro Ala Val Glu   1 5 10 15 Gly Arg Ala Thr Ile              20 <210> 44 <211> 21 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H2 <400> 44 Ser Ile Thr Ser Gly Gly Ser Gly Thr Trp Asn Ala Pro Ala Val Lys   1 5 10 15 Gly Arg Ala Thr Ile              20 <210> 45 <211> 21 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H2 <400> 45 Ser Ile Thr Ser Gly Gly Ser Gly Thr Trp Tyr Ala Pro Ala Val Lys   1 5 10 15 Gly Arg Ala Ser Ile              20 <210> 46 <211> 21 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H2 <400> 46 Ser Ile Thr Ser Gly Gly Ser Gly Thr Trp Tyr Ala Pro Thr Val Lys   1 5 10 15 Gly Arg Ala Thr Ile              20 <210> 47 <211> 21 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H2 <400> 47 Ser Ile Thr Ser Gly Gly Ser Gly Thr Trp Tyr Ala Ser Thr Val Lys   1 5 10 15 Gly Arg Ala Thr Ile              20 <210> 48 <211> 21 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H2 <400> 48 Ser Ile Thr Ser Gly Gly Ser Gly Thr Trp Tyr Ala Pro Ala Val Lys   1 5 10 15 Gly Arg Ala Ile Ile              20 <210> 49 <211> 13 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H3 <400> 49 Ser Ala Tyr Gly Gly Trp Ile Ala Asp Asp Ile Asp Ala   1 5 10 <210> 50 <211> 13 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H3 <400> 50 Ser Ala Tyr Gly Gly Trp Ile Ala Glu Asp Ile Asp Ala   1 5 10 <210> 51 <211> 13 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H3 <400> 51 Ser Val Tyr Gly Gly Trp Ile Ala Asp Asp Ile Asp Ala   1 5 10 <210> 52 <211> 13 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H3 <400> 52 Ser Ala Tyr Gly Gly Trp Ile Ala Asp Asp Val Asp Ala   1 5 10 <210> 53 <211> 13 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H3 <400> 53 Ser Ala Tyr Gly Gly Trp Ile Ala Ser Asp Ile Asp Ala   1 5 10 <210> 54 <211> 13 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H3 <400> 54 Ser Ala Tyr Gly Gly Trp Ile Ala Asp Asp Ile Asp Ser   1 5 10 <210> 55 <211> 13 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H3 <400> 55 Ser Ala Tyr Gly Gly Trp Ile Ala Asp Asp Ile Asp Val   1 5 10 <210> 56 <211> 13 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H3 <400> 56 Ser Ala Tyr Gly Gly Trp Ile Ala Asp Asp Thr Asp Ala   1 5 10 <210> 57 <211> 13 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H3 <400> 57 Ser Ala Tyr Gly Gly Trp Ile Ala Asp Asp Ile Val Ala   1 5 10 <210> 58 <211> 13 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H3 <400> 58 Ser Ala Tyr Gly Gly Trp Ile Ala Asp Asp Ile Asp Thr   1 5 10 <210> 59 <211> 13 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H3 <400> 59 Ser Ala Tyr Gly Gly Trp Ile Ala Asp Asp Ile Asn Ala   1 5 10 <210> 60 <211> 13 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H3 <400> 60 Ser Ala Tyr Gly Gly Trp Ile Val Asp Asp Ile Asp Ala   1 5 10 <210> 61 <211> 13 <212> PRT <213> Anti-AFP-L3 antibody heavy chain CDR-H3 <400> 61 Ser Ala Tyr Gly Gly Trp Ile Ala Gly Asp Ile Asn Ala   1 5 10 <210> 62 <211> 7 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L1 <400> 62 Ser Gly Gly Ser Gly Tyr Gly   1 5 <210> 63 <211> 7 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L1 <400> 63 Ser Gly Gly Ser Asp Tyr Gly   1 5 <210> 64 <211> 7 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L1 <400> 64 Ser Gly Gly Ser Ser Tyr Gly   1 5 <210> 65 <211> 7 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L1 <400> 65 Ser Gly Gly Ser Gly Cys Gly   1 5 <210> 66 <211> 7 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L1 <400> 66 Ser Gly Gly Arg Ser Tyr Gly   1 5 <210> 67 <211> 7 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L1 <400> 67 Ser Gly Gly Ser Gly His Gly   1 5 <210> 68 <211> 7 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L1 <400> 68 Ser Gly Gly Arg Gly Tyr Gly   1 5 <210> 69 <211> 8 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L2 <400> 69 Tyr Glu Ser Asn Lys Arg Pro Ser   1 5 <210> 70 <211> 8 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L2 <400> 70 Tyr Glu Ser Asp Lys Arg Pro Ser   1 5 <210> 71 <211> 8 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L2 <400> 71 Tyr Glu Ser Thr Lys Arg Pro Ser   1 5 <210> 72 <211> 8 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L2 <400> 72 Tyr Glu Ser Ser Lys Arg Pro Ser   1 5 <210> 73 <211> 8 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L2 <400> 73 Tyr Glu Ser Asn Met Arg Pro Ser   1 5 <210> 74 <211> 8 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L2 <400> 74 Tyr Glu Ser Asn Arg Arg Pro Ser   1 5 <210> 75 <211> 8 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L2 <400> 75 Tyr Glu Ser Thr Lys Arg Pro Pro   1 5 <210> 76 <211> 8 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L2 <400> 76 Tyr Glu Asn Asn Lys Arg Pro Ser   1 5 <210> 77 <211> 8 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L2 <400> 77 Tyr Glu Ser His Lys Arg Pro Ser   1 5 <210> 78 <211> 8 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L2 <400> 78 Tyr Glu Ser Asn Lys Arg Pro Pro   1 5 <210> 79 <211> 8 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L2 <400> 79 Tyr Glu Ser Asn Lys Arg Pro Thr   1 5 <210> 80 <211> 8 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L2 <400> 80 Tyr Glu Ser Asn Lys Arg Leu Ser   1 5 <210> 81 <211> 8 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L2 <400> 81 Tyr Glu Ser Asn Lys Arg Pro Leu   1 5 <210> 82 <211> 8 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L2 <400> 82 Tyr Asp Ser Asn Lys Arg Pro Ser   1 5 <210> 83 <211> 8 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L2 <400> 83 Tyr Asp Ser Asp Lys Arg Pro Ser   1 5 <210> 84 <211> 8 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L2 <400> 84 Tyr Glu Cys Asn Lys Arg Pro Ser   1 5 <210> 85 <211> 8 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L2 <400> 85 Tyr Glu Ser Tyr Lys Arg Pro Ser   1 5 <210> 86 <211> 14 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L3 <400> 86 Gly Gly Trp Glu Ser Ser Ser Asn Pro Gly Ile Phe Gly Ala   1 5 10 <210> 87 <211> 14 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L3 <400> 87 Gly Gly Arg Glu Ser Ser Ser Asn Pro Gly Ile Phe Gly Ala   1 5 10 <210> 88 <211> 14 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L3 <400> 88 Gly Gly Trp Glu Ser Asn Ser Asn Pro Gly Ile Phe Gly Ala   1 5 10 <210> 89 <211> 14 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L3 <400> 89 Gly Gly Trp Gly Ser Ser Ser Asn Pro Gly Ile Phe Gly Ala   1 5 10 <210> 90 <211> 14 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L3 <400> 90 Gly Gly Trp Glu Ser Asn Ser Asn Pro Gly Thr Phe Gly Ala   1 5 10 <210> 91 <211> 14 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L3 <400> 91 Gly Gly Trp Glu Ser Ser Asn Pro Gly Val Phe Gly Ala   1 5 10 <210> 92 <211> 14 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L3 <400> 92 Gly Gly Trp Glu Ser Gly Ser Asn Pro Gly Ile Phe Gly Ala   1 5 10 <210> 93 <211> 14 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L3 <400> 93 Gly Gly Trp Glu Ser Ser Ser Asn Pro Gly Ile Phe Gly Thr   1 5 10 <210> 94 <211> 14 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L3 <400> 94 Gly Gly Trp Glu Ser Tyr Ser Asn Pro Gly Ile Phe Gly Ala   1 5 10 <210> 95 <211> 14 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L3 <400> 95 Gly Gly Trp Glu Ser Val Ser Asn Pro Gly Ile Phe Gly Ala   1 5 10 <210> 96 <211> 14 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L3 <400> 96 Gly Gly Trp Glu Ser Ser Ser Ser Pro Gly Ile Phe Gly Ala   1 5 10 <210> 97 <211> 14 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L3 <400> 97 Gly Gly Trp Glu Ser Ser Asn Asn Pro Gly Ile Phe Gly Ala   1 5 10 <210> 98 <211> 14 <212> PRT <213> Anti-AFP-L3 antibody light chain CDR-L3 <400> 98 Gly Gly Trp Glu Ser Ser Ser Lys Pro Gly Ile Phe Gly Ala   1 5 10 <210> 99 <211> 72 <212> DNA <213> VH forward primer <400> 99 ggtcagtcct ctagatcttc cggcggtggt ggcagctccg gtggtggcgg ttccgccgtg 60 acgttggacg ag 72 <210> 100 <211> 44 <212> DNA <213> VH reverse primer <400> 100 ctggccggcc tggccactag tggaggagac gatgacttcg gtcc 44 <210> 101 <211> 38 <212> DNA <213> VL forward primer <400> 101 gtggcccagg cggccctgac tcagccgtcc tcggtgtc 38 <210> 102 <211> 34 <212> DNA <213> VL reverse primer <400> 102 ggaagatcta gaggactgac ctaggacggt cagg 34 <210> 103 <211> 42 <212> DNA <213> scFV forward primer <400> 103 gaggaggagg aggaggaggt ggcccaggcg gccctgactc ag 42 <210> 104 <211> 47 <212> DNA <213> scFV reverse primer <400> 104 gaggaggagg aggaggagga gctggccggc ctggccacta gtggagg 47 <210> 105 <211> 20 <212> DNA <213> Seqeuncing primer (forward) <400> 105 acactttatg cttccggctc 20 <210> 106 <211> 20 <212> DNA <213> Seqeuncing primer (reverse) <400> 106 caaaatcacc ggaaccagag 20 <210> 107 <211> 19 <212> DNA <213> VH primer (forward) <400> 107 gctagccgcc accatgggc 19 <210> 108 <211> 37 <212> DNA <213> VH primer (reverse) <400> 108 aggggccctt ggtggaggcc tggccggcct ggccact 37 <210> 109 <211> 20 <212> DNA <213> CH primer (forward) <400> 109 gcctccacca agggcccctc 20 <210> 110 <211> 20 <212> DNA <213> CH primer (reverse) <400> 110 cgggatccct tgccggccgt 20 <210> 111 <211> 19 <212> DNA <213> HC primer (forward) <400> 111 gctagccgcc accatgggc 19 <210> 112 <211> 20 <212> DNA <213> HC primer (reverse) <400> 112 cgggatccct tgccggccgt 20 <210> 113 <211> 18 <212> DNA <213> VL primer (forward) <400> 113 aagcttgccg ccaccatg 18 <210> 114 <211> 37 <212> PRT <213> VL primer (reverse) <400> 114 Ala Gly Gly Gly Gly Gly Cys Gly Gly Cys Cys Ala Cys Gly Gly Thr   1 5 10 15 Cys Cys Gly Gly Aly Gly Aly Gly Aly Thr Cys Thr Ala Gly Aly Gly              20 25 30 Gly Ala Cys Thr Gly          35 <210> 115 <211> 20 <212> DNA <213> Ck primer (forward) <400> 115 cggaccgtgg ccgccccctc 20 <210> 116 <211> 19 <212> DNA <213> Ck primer (reverse) <400> 116 gctctagact agcactcgc 19 <210> 117 <211> 18 <212> DNA <213> LC primer (forward) <400> 117 aagcttgccg ccaccatg 18 <210> 118 <211> 19 <212> DNA <213> LC primer (reverse) <400> 118 gctctagact agcactcgc 19

Claims (17)

At least one heavy chain variable region complementary binding site selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and a heavy chain variable region complementary binding region (CDR) And at least one light chain variable region complementary binding site selected from the group consisting of light chain variable region complementary binding regions (CDRs) each consisting of the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 6, and specifically binding to alpha-fetoprotein L3 Or an antigen-binding fragment thereof. The antibody of claim 1, wherein the antibody comprises a heavy chain variable region complementary binding region (CDR) comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, Wherein the antibody or antigen-binding fragment thereof specifically binds to alpha-fetoprotein L3, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region complementary binding site (CDR). The antibody or an antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof specifically binds to AFP-L3 in a manner distinct from AFP-L1. The anti-AFP-L3 antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof has a ratio of the AFP-L3 O.D value to AFP-L1 of 1.5 or more. The antibody or its antigen-binding fragment thereof according to claim 1, wherein the concentration of AFP-L3 is 50 ng / ml or more. The method of claim 1, wherein the heavy chain variable region complementary binding site comprises CDR-H1 selected from the group consisting of the amino acid sequences of SEQ ID NOS: 7 to 27, CDR-H2 selected from the group consisting of amino acid sequences of SEQ ID NOS: 28 to 48, An anti-AFP-L3 antibody comprising CDR-H3 selected from the group consisting of the amino acid sequences of SEQ ID NOS: 49 to 61, or an antigen-binding fragment thereof. The light chain variable region complementary binding site according to claim 1, wherein the light chain variable region complementary binding site comprises CDR-L1 selected from the group consisting of the amino acid sequences of SEQ ID NOS: 62 to 68, CDR-L2 selected from the group consisting of amino acid sequences of SEQ ID NOS: 69 to 85, An anti-AFP-L3 antibody comprising CDR-L3 selected from the group consisting of the amino acid sequences of SEQ ID NOS: 86-98, or an antigen-binding fragment thereof. The anti-AFP-L3 antibody or antigen-binding fragment thereof according to claim 1, wherein said anti-AFP-L3 antibody is a monoclonal antibody. The anti-AFP-L3 antibody or antigen-binding fragment thereof according to claim 1, wherein said anti-AFP-L3 antibody is a mouse-derived antibody, mouse-human chimeric antibody, humanized antibody or human antibody. The anti-AFP-L3 antibody of claim 1, wherein said antigen binding fragment is selected from the group consisting of scFv, (scFv) ₂ , Fab, Fab 'and F (ab') ₂ of said anti-AFP- Or an antigen-binding fragment thereof. A composition for the diagnosis of an AFP-L3 related disease, comprising the anti-AFP-L3 antibody according to any one of claims 1 to 10 or an antigen-binding fragment thereof. 12. The composition of claim 11, wherein the composition further comprises an anti-AFP antibody that differs in antibody-binding site from the antibody to core-fucosylated AFP. 12. The composition of claim 11, further comprising a marker conjugated directly or via a linker to the anti-AFP antibody. 12. The diagnostic composition of claim 11, wherein the disease is liver cancer. 11. A method for detecting AFP-L3 antibodies comprising contacting an anti-AFP-L3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 with a sample, and detecting the binding reaction of the anti- -L3. &Lt; / RTI &gt; 16. The method of claim 15, wherein the method comprises the steps of: ELISA, radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA) or luminescence immunoassay (LIA) / RTI &gt; The anti-AFP-L3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 as a capture antibody, which is different from the anti-core-fucosylated AFP antibody as a detection antibody An anti-AFP antibody, and a marker conjugated to the detection antibody directly or via a linker.

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