JP2006055141A - Method for identifying antibody specific to sugar chain structure - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for identifying an antibody specifically bonding with a sugar chain having a certain specific structure by using a phage presentation type antibody library and the sugar chain. <P>SOLUTION: This method for identifying the antibody bonding specifically with the sugar chain comprises a process of bringing the phage presentation type antibody in contact with the sugar chain. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for identifying a sugar-binding antibody. More specifically, the present invention relates to a method for identifying an antibody that specifically binds to a sugar chain by analyzing the binding between a phage-displayed antibody library and a solid phase-immobilized sugar chain.

  Complex carbohydrate molecules or cell surface sugar chains are involved in various life phenomena. In particular, from the knowledge that the structure or distribution of sugar chains changes with diseases such as cancer, it is expected that investigation of sugar chains will lead to the elucidation of the cause of disease and the mechanism of progression. Therefore, it is important to detect specific sugar chains distributed in molecules or tissues simply and with high accuracy.

  Conventionally, in order to detect specific sugar chains in tissues or molecules, lectins and antibodies that recognize target sugar chains have been used. However, the specificity of lectins for sugars is low, and it is not suitable for detailed analysis of sugar chain structures, such as identifying the binding mode of sugar residues and oligosaccharide structures. Moreover, although the antibody produced by the immunological method has high specificity, the antibody with respect to the sugar chain which has antigenicity cannot be obtained, and the kind is also limited.

  On the other hand, since sugar-binding phage-displaying antibodies using the phage display method are produced without using immunological techniques, many types of antibodies can be obtained regardless of immunogenicity. It is expected to be able to identify sugar chains with high specificity and complex structure.

  An object of the present invention is to provide a method for identifying an antibody that specifically binds to a sugar chain having a specific structure using a phage-displayed antibody library and a sugar chain. Furthermore, another object of the present invention is to provide an antibody and an antibody library obtained by the above method.

  As a result of intensive studies to solve the above problems, the present inventors have selected an antibody that specifically binds to the sugar chain by bringing a phage-displayed antibody into contact with a sugar chain having a specific structure. The present invention has been completed by finding that it can be amplified and identified automatically.

  That is, according to the present invention, there is provided a method for identifying an antibody that specifically binds to a sugar chain, which comprises bringing a phage-displayed antibody into contact with the sugar chain.

Preferably, the sugar chain is immobilized on a solid phase.
Preferably, the sugar chain is a glycolipid, more preferably an artificial glycolipid.
Preferably, as the sugar component constituting the sugar chain, for example, D-mannose (D-Man), L-fucose (L-Fuc), DN-acetylglucosamine (D-GlcNAc), D-glucose (D -Glc), D-galactose (D-Gal), or DN-acetylgalactosamine (D-GalNAc).

Preferably, the phage-displayed antibody is a human-type single chain phage library.
Preferably, the phage-displayed antibody is obtained by amplifying a fragment containing the CDR1 and CDR2 regions of the VH or VL region of an immunoglobulin gene and a fragment containing the CDR3 region by PCR using a human cDNA library as a template, As a template, VH or VL region is amplified by PCR method, and these amplified DNA fragments are respectively incorporated into non-expression type phagemid vectors to prepare VH library and VL library. A VL library is infected with helper phage, converted into a phage library, and these phage-type VH and VL libraries are co-infected with Escherichia coli expressing Cre recombinase to recombine between VH and VL vectors in E. coli. The helper phage Is obtained by generating phage expressing full-length single chain antibody were infected.

  Preferably, the phage-displayed antibody is recovered from a human-type single-chain phage library using as an index the ability to bind to an artificial glycolipid having a predetermined sugar chain structure.

  Preferably, the method of the present invention prepares a human-type single-stranded phage library and binds to the sugar chain structure using the binding ability to an artificial glycolipid having a predetermined sugar chain structure as an index. It is a method comprising obtaining a phage by panning and contacting the obtained human single-stranded phage with a sugar chain immobilized on a solid phase.

Preferably, the binding between the sugar chain and the phage-displayed antibody is detected using an anti-phage antibody labeled with a detectable substance.
Preferably, the binding between the sugar chain and the phage-displayed antibody is detected by ELISA.

  According to another aspect of the present invention, there is provided an antibody or antibody library that specifically binds to a specific sugar chain, which is isolated by the above-described method of the present invention.

According to still another aspect of the present invention, there is provided an antibody having an amino acid sequence set forth in SEQ ID NOs: 1 and 2, which has a binding ability specific to M3-DPPE described below. Examples of the antibody of the present invention are antibodies having the amino acid sequences set forth in SEQ ID NOs: 3 and 4.
According to still another aspect of the present invention, a DNA having a base sequence encoding the amino acid sequence described in SEQ ID NOs: 1 and 2 and a DNA having a base sequence encoding the amino acid sequence described in SEQ ID NOs: 3 and 4 Provided.

  According to the present invention, it is possible to obtain antibodies specific to various sugar chains that have been impossible to isolate so far, and thus it is possible to construct an antibody library. The methods of the present invention are essentially different from conventional methods of immunizing individuals to produce polyclonal or monoclonal antibodies. That is, in the present invention, antibodies that specifically bind with high affinity to various sugar chains including sugar chain antigens that cannot be recognized by individuals are isolated by utilizing a phage display method that can achieve the antibody production mechanism in vitro. can do. Furthermore, improved secondary single chain antibodies can be produced by genetic manipulation of single chain antibodies obtained by the method of the present invention, and mass production of single chain antibodies is easy. Furthermore, sugar chain-specific delivery of proteins such as toxins and enzymes is also possible by genetic manipulation of the antibodies obtained in the present invention.

Hereinafter, embodiments of the present invention will be described more specifically.
(1) Artificial glycolipid As the glycolipid used in the present invention, for example, an artificial glycolipid described in Japanese Patent No. 2828391 can be used. Although the kind of saccharide | sugar component which comprises an oligosaccharide is not specifically limited, For example, D-mannose (D-Man), L-fucose (L-Fuc), DN-acetylglucosamine (D-GlcNAc), D-glucose Examples include monosaccharides such as (D-Glc), D-galactose (D-Gal), and DN-acetylgalactosamine (D-GalNAc).

  Furthermore, ganglioside, which is a glycosphingolipid having a sialic acid residue, can also be used. Gangliosides are known to exist on the cell membrane surface and regulate cell proliferation, differentiation, or signal transduction. Gangliosides interact with other cells and tissues in cancer cell invasion and metastasis. It is known to be involved in the action process.

  In the oligosaccharide, each constituent saccharide is bonded by α1 → 2 bond, α1 → 3 bond, α1 → 4 bond, α1 → 6 bond, β1 → 4 bond, or the like, or a combination thereof. For example, mannose may form a straight chain by the above bond, or may have a branched structure by a combination of α1 → 3 bond and α1 → 6 bond. The number of monosaccharides in the oligosaccharide is preferably 2 to 11.

  Specific examples of oligosaccharides include mannobiose (M2), mannotriose (M3), mannotetraose (M4), mannopentaose (M5), mannohexaose (M6), and mannoheptaose (M7). ), Various mixed oligosaccharides such as M5 (Chemical Formula 1) and RN (Chemical Formula 2) shown below.

  Furthermore, as oligosaccharides containing glucose, those having the structure shown in Chemical Formula 3 can be mentioned, and as oligosaccharides containing N-acetylglucosamine, those shown in Chemical Formula 4 can be mentioned, and as oligosaccharides containing fucose The thing shown to Chemical formula 5 can be mentioned.

These oligosaccharides all have one reducing terminal aldehyde group. Therefore, this aldehyde group can be used as a means for immobilizing oligosaccharides. That is, a Schiff base is formed by a reaction between the aldehyde group and a lipid having an amino group, and then the Schiff base is reduced according to a conventional method, preferably a chemical reduction, for example, NaBH ₃ CN is used to convert an oligosaccharide and a lipid. Can be combined (Tetsuo Mizuochi, Munehiro Nakata, Glycobiology Experimental Protocol, pages 42-47, Shujunsha, 1996).

  The lipid having an amino group is preferably a phospholipid having an amino group, and for example, dipalmitoyl phosphatidylethanolamine (DPPE), distearoyl phosphatidylethanolamine (DSPE) and the like can be used. In the present invention, the conjugate of oligosaccharide and lipid obtained as described above may be referred to as artificial glycolipid.

  The glycolipid used in the present invention may be the above-mentioned artificial glycolipid, and further, natural products such as glycolipids obtained from bovine brain, cancer cells, or other animals and plants, and these glycolipids are modified. For example, a product to which a glycosyltransferase is allowed to act to add a sugar chain or a chemically synthesized product can be used. Specific examples include gangliosides (GT1a, GD1a, GM1, GM2, GM3, GM4, GQ1b, GT1b, GD1b, GD2, GD3, GP1c, GQ1c, GT1c, GT2 and GT3).

(2) Antigen-coated plate The solvent for dissolving the artificial glycolipid serving as the antigen is not particularly limited, but after dissolving in chloroform / methanol / water (10: 10: 3, volume ratio), further with methanol, for example, 40 μg / ml It is desirable to dilute. This solution is added to, for example, a 96-well plastic plate (flat bottom) and air-dried to fix the artificial glycolipid to the well. The well is blocked and washed. A plate coated with artificial glycolipid by such an operation may be referred to as an antigen-coated plate.

It is desirable to use Tris buffered saline (hereinafter referred to as TBS) as the buffer solution. The blocking solution includes, for example, TBS containing 3% bovine serum albumin (hereinafter referred to as 3% BSA / TBS), and the cleaning solution includes, for example, TBS including 0.2% Tween 20 (hereinafter referred to as TBS-T). desirable.

(3) Phage-displayed antibodies Phage-displayed antibodies can be prepared by known methods (Marks JD et al., J. Mol. Biol. 222, 581-597, 1991; Nissim A et al., EMBO J. et al. 13: 692-698, 1994; Takayanagi Satoshi, Okui Riyo, Shimizu Nobuyoshi, Super Repertoire Artificial Antibody Library, Application No. Japanese Patent Application No. 2001-358602 (International Publication No. WO 03/04419)).

  The phage-displayed antibody library is an application of a system that displays (displays) an antibody on the surface of a phage by fusing the antibody to a coat protein of a filamentous phage. Specifically, a phage display library can be obtained by amplifying antibody genes by PCR to prepare a library containing many types of antibody genes and displaying them on phages. Although the specific example of the preparation method of a phage display type | mold single chain antibody is shown below, it is not limited to this.

  Using a human peripheral blood and spleen cDNA library as a template, a fragment containing the CDR1 and CDR2 regions of the VH or VL region of an immunoglobulin gene and a fragment containing the CDR3 region are each amplified by PCR. These are mixed and used as a template to amplify the VH or VL region by PCR. Each of these amplified DNA fragments is incorporated into a non-expression type phagemid vector to prepare a VH library and a VL library. These E. coli VH libraries and VL libraries are infected with helper phages and converted into phage libraries. These phage type VH and VL libraries are co-infected with Escherichia coli expressing Cre recombinase, and a recombination reaction is carried out between the VH / VL vectors in E. coli. This E. coli can be infected with M13KO7 helper phage to generate a phage that expresses a full-length single chain antibody. In order to remove non-recombinants contaminated with these phages, this phage is infected with E. coli so that multiple infections do not occur, and further helper phages are superinfected, and glucose containing ampicillin, kana machine, chloramphenicol Incubate at 25 ° C. in a medium containing no Thereby, a phage expressing a recombinant single chain antibody in the supernatant can be obtained. The phage in the supernatant can be precipitated with polyethylene glycol and resuspended to obtain an artificial antibody library having a repertoire of 10 11 or more.

  Furthermore, the phage-displaying antibody having the target sugar-binding property is selectively recovered from the human-type single-stranded phage library prepared by the above method using the binding ability to the artificial glycolipid having the sugar chain structure as an index. be able to. This operation may be referred to as panning.

(4) Sugar-binding analysis method of phage-displayed antibody As a method of analyzing the sugar-binding property of a phage-displayed antibody obtained by repeating panning, for example, analysis by applying the ELISA method described below can be performed. It is desirable because a large number of phage clone samples can be analyzed simultaneously. That is, a sugar chain is immobilized on a 96-well plastic plate and blocked with, for example, a buffer containing 3% BSA in order to eliminate nonspecific adsorption of the phage-displayed antibody. To this, a suspension of a phage-displayed antibody prepared to an appropriate concentration with a buffer solution is added and allowed to react sufficiently (for example, at 37 ° C. for 2 hours), and then washed with a buffer solution.

  In order to detect and quantify phage-displayed antibodies bound to sugar chains, react with an aqueous solution of anti-phage antibody labeled with a substance suitable for detection, wash with a buffer solution, and then use the optimal method for detection of the labeled substance. Detect and quantify. At this time, the labeling substance of the anti-phage antibody is not particularly limited, but for example, labeling with horseradish peroxidase is desirable in terms of simplicity. The detector used for detection is not particularly limited, but it is desirable to use, for example, a plate reader suitable for ELISA.

  The following examples further illustrate the present invention, but the present invention is not limited to the examples.

Example 1: Phage-displaying single-chain antibody having binding ability to mannotriose or lacto-N-fucopentaose III (1) Preparation of artificial glycolipid First, mannotriose (M3) or lacto-N-fucopentaose III (
LNFPIII) was dissolved in 2.5 mg of distilled water by adding 600 μl of distilled water to prepare an oligosaccharide solution. Next, DPPE was dissolved in a chloroform / methanol (1: 1, volume ratio) mixture to a concentration of 5 mg / ml to prepare a DPPE solution. Further, a NaBH ₃ CN solution was prepared by dissolving NaBH ₃ CN in methanol at a concentration of 10 mg / ml. To 600 μl of each oligosaccharide solution, 9.4 ml of the DPPE solution and 1 ml of the NaBH ₃ CN solution were added and mixed. This mixed solution was kept at 60 ° C. for 16 hours to produce an artificial glycolipid.

  The artificial glycolipid synthesized as described above was purified by high performance liquid chromatography (HPLC) using silica gel column and ODS column. A silica gel column was used at room temperature using Shim-pack PREP-SIL. After equilibrating the column with chloroform / methanol / 50 mM acetic acid (65: 30: 5, volume ratio), a sample containing an artificial glycolipid dissolved in the same solvent was injected, and the ratio of chloroform, methanol and 50 mM acetic acid in 60 minutes. A linear gradient elution was performed with a ratio of 50:55:18. The flow rate was 2 ml / min, and the eluate was fractionated by 4 ml. The ODS column was equilibrated with 50 mM acetic acid, a sample containing artificial glycolipid dissolved in the same solvent was added to the column, the column was further washed with the same solvent, and then 50% acetonitrile, methanol, and chloroform / methanol / 50 mM acetic acid. (105: 100: 28, volume ratio) was sequentially flowed to elute the artificial glycolipid.

(2) Preparation of antigen-coated plate and phage recovery plate Artificial glycolipid was dissolved in chloroform / methanol / water (10: 10: 3, volume ratio) at a concentration of 1 mg / ml, and further diluted 25-fold with methanol ( Final concentration 40 μg / ml). This solution is placed in a 96-well plastic plate (flat bottom) at 50 μl per well (2 μg / well), allowed to stand at 37 ° C. and air-dried. Add 3% BSA / TBS to the well and allow to stand at 4 ° C. overnight to block. This is washed twice with TBS-T and then once with TBS.

  Meanwhile, 150 μl of 3% BSA / TBS is placed in another 96-well plastic plate, left to stand at 4 ° C. overnight, and washed twice with TBS-T and once with TBS. This plate may be referred to as a phage collection plate.

(3) Preparation method of antibody library Using human peripheral blood and spleen cDNA library as a template, a fragment containing CDR1 and CDR2 regions of immunoglobulin gene VH or VL region and a fragment containing CDR3 region are amplified by PCR method, respectively. did. VH or VL region was amplified by PCR method using them as a template.

  These amplified DNA fragments were each incorporated into a non-expression type phagemid vector to prepare a VH library and a VL library. Each was prepared so as to contain 10 6 and 10 5 or more colonies. These E. coli VH library and VL library were infected with M13KO7 helper phage and converted to phage libraries. These phage-type VH and VL libraries were co-infected with Escherichia coli expressing Cre recombinase, and a recombination reaction was carried out between the VH / VL vectors in E. coli. This E. coli was infected with M13KO7 helper phage to generate a phage expressing a full-length single chain antibody. In order to remove non-recombinants contaminated with these phages, this phage was infected with E. coli XL1-Blue strain so that multiple infection would not occur, and further M13KO7 helper phage was superinfected, and ampicillin, kana machine, chloram The cells were cultured at 25 ° C. in a medium containing phenicol and no glucose. As a result, a phage expressing the recombinant single chain antibody in the supernatant was obtained. The phage in the supernatant was precipitated with polyethylene glycol and resuspended to obtain an artificial antibody library having a repertoire of 10 11 or more. It was stored frozen at −80 ° C. until use.

(4) Panning operation A 250 μl human single-stranded phage library (1.8 × 10 ¹⁵ pfu / ml) contains 1.3 ml of TBS, 1.3 ml of 3% BSA / TBS and 30 μl of 10% Tween20. TBS was mixed and incubated at 37 ° C. for 1 hour.

  To the antigen-coated plate, 50 μl of the phage library treated as described above is added per well and incubated at 37 ° C. for 1 hour. The well was washed 3 times with TBS-T and twice with TBS, and then the remaining liquid was carefully removed. 50 μl of 100 mM triethylamine (hereinafter referred to as TEA) was added to each well and allowed to stand at room temperature for 10 minutes to release bound phages.

  On the other hand, a neutralization solution (hereinafter referred to as NR) prepared by mixing 1M Tris-HCl buffer (pH 7.4) and 3% BSA / TBS at a ratio of 2: 1 was added to a phage recovery plate at 100 μl per well. added. In this well, TEA containing the phage released by the above operation was recovered. Again, TEA was added to the wells of the antigen-coated plate and allowed to stand at room temperature for 20 minutes, and then recovered in NR.

  To the collected phage solution, 100 μl of E. coli TG-1 suspension was added per well and incubated at 37 ° C. for 1 hour. This was seeded on an LBGC (LB / glucose / carbenicillin) agar plate and cultured at 25 ° C. to form colonies. Colonies were collected and rotated (210 rpm) for 2 hours in SBS medium containing carbenicillin. Next, helper phages were added and allowed to stand at 37 ° C. for 1 hour, and then kanamycin and chloramphenicol were added, followed by rotary culture (210 rpm) at 25 ° C. for 2 nights.

  The culture solution was centrifuged (5000 × g, 30 minutes, 4 ° C.), 1/4 amount of 20% polyethylene glycol / 2.5 M NaCl solution was added to the supernatant, and the mixture was allowed to stand in ice for 1 hour. After centrifugation (15000 × g, 30 minutes, 4 ° C.), the precipitated phage was suspended in TBS and collected. To this was added TBS containing an equal amount of 3% BSA / TBS and 1/20 amount of 10% Tween 20, and kept at 37 ° C. for 1 hour, followed by centrifugation to remove insoluble components (18000 × g, 5 minutes, 4 ° C.) The supernatant was recovered as primary panning phage.

  For the recovered phage, the same panning operation as described above was repeated to recover the quaternary panning phage. However, a 24-well plate was used in the secondary panning operation, a 12-well plate was used in the tertiary panning operation, and a 6-well plate was used in the fourth panning operation. Further, in the washing operation after the third panning, in addition to washing with TBS-T and TBS, washing with 100 mM glycine hydrochloride buffer (pH 2.7) was also performed.

  Panning using an artificial glycolipid prepared from mannotriose (hereinafter referred to as M3-DPPE) or an artificial glycolipid prepared from lacto-N-fucopentaose III (hereinafter referred to as LNFPIII-DPPE) according to the above method, About each phage clone obtained by the fourth panning, the binding property to the artificial glycolipid was examined by the ELISA method described below.

(5) ELISA
SBS medium containing carbenicillin was placed in a 96-well plastic plate (round bottom) at 50 μl per well, and quaternary panning phage colonies were transferred to this and cultured at 37 ° C. for 1 hour. Next, after adding helper phage and rotating culture at 37 ° C. for 1 hour, an SBS medium containing kanamycin and chloramphenicol was added, followed by rotating culture at 25 ° C. overnight. 100 μl of 3% BSA / TBS was added to 50 μl of the supernatant obtained by centrifugation (1500 rpm, 15 minutes, 4 ° C.) and incubated at 37 ° C. for 1 hour.

  After immobilizing 1 μg of artificial glycolipid (M3-DPPE or LNFPIII-DPPE) in each well of a 96-well plastic plate, it was blocked overnight with 150 μl of 3% BSA / TBS at 4 ° C. In addition, a well not coated with artificial glycolipid was also prepared as a control. To this was added 75 μl of the above phage solution and incubated at 37 ° C. for 1 hour. As a control, the same operation was performed for wells to which no antigen was immobilized. The wells were washed 5 times with 200 μl of TBS-T, 50 μl of horseradish peroxidase (hereinafter referred to as HRP) -labeled anti-M13 antibody solution was added, and the mixture was incubated at 37 ° C. for 1 hour. The wells were washed 10 times with 200 μl TBS-T and then once with 200 μl TBS. 100 μl of HRP coloring solution was added to this and reacted at room temperature. After 30 minutes, 100 μl of 2% oxalic acid was added to stop the reaction. This was set in a plate reader and the absorption at 415 nm was measured.

  The measurement results are shown in FIGS. FIG. 1 shows an example of examining the binding of each phage clone panned with M3-DPPE to M3-DPPE, and FIG. 2 shows the binding of each phage clone panned with LNFPIII-DPPE to LNFPIII-DPPE. This is an example of examining sex. In the graphs of FIG. 1 and FIG. 2, colonies that had an absorbance ratio of at least twice when the artificial glycolipid was coated ((+) in the figure) and when not coated ((-) in the figure) were positive clones. It was.

Example 2: Cloning and sequencing of positive clones (1) Colony PCR and restriction enzyme treatment The following primers were used.
cmf: 5'-TGTGATGGCTTCCATGTCGGCAGAATGC-3 '(SEQ ID NO: 5)
g3-R: 5'-GCTAAACAACTTTCAACTTTCAATAGTCTATGGGGCAC-3 '(SEQ ID NO: 6)

The operation is as follows. Antigen positive antibody phage is infected with E. coli (TG-1 strain or XL-1 blue strain) and seeded on an agar medium. In the case of TG-1 strain, carbenicillin-containing LBGC agar medium is used, and in the case of XL-1 blue strain, tetracycline and ampicillin-containing LBGTA agar medium is used. Culture at 25 ° C. for 1-2 days to form colonies. Reaction in which 4 types of deoxynucleotide triphosphates (dNTPs) 0.2 mM each, the above two types of primers 0.5 μM each, and Taq polymerase 2.5 U / 100 μl added to a PCR buffer containing MgCl ₂ Prepare a solution and place 20 μl in a PCR tube. Pick up the colony with the insulator and put it in the reaction mixture and shake the insulator several times. After mounting on a PCR machine and pre-incubating at 94 ° C. for 2 minutes, the cycle of 94 ° C. for 20 seconds, 50 ° C. for 10 seconds and 68 ° C. for 1 minute is repeated 30 cycles, and finally stored at 4 ° C. A 1% agarose gel is prepared, and the PCR sample is subjected to agarose gel electrophoresis to confirm the presence and size of the PCR product.

MvaI was used for the restriction enzyme treatment. The operation is as follows. Add to TA buffer and incubate at 37 ° C for 1 hour. Electrophoresis is performed on a 4% agarose gel (2% standard agarose, 2% low-melting agarose), and the cleavage patterns are compared for each clone.

(2) scFv sequencing Phage clones M3A4-12 and M3G4-10 capable of binding to M3-DPPE were sequenced. The results are shown in FIGS. The amino acid sequence of VH (and the base sequence encoding it) of phage clone M3A4-12 is SEQ ID NO: 1, the amino acid sequence of VL (and the base sequence encoding it) is SEQ ID NO: 2, and the VH of phage clone M3G4-10. The amino acid sequence (and the base sequence encoding it) is shown in SEQ ID NO: 3, and the amino acid sequence of VL (and the base sequence encoding it) is shown in SEQ ID NO: 4.

FIG. 1 is an example of examining the binding of M3-DPPE to the obtained phage clone (24 clones) panned with M3-DPPE, and 4 clones out of 24 clones are positive. FIG. 2 is an example of examining the binding of the obtained phage clone (24 clones) panned with LNFPIII-DPPE to LNFPIII-DPPE, and 23 clones out of 24 clones are positive. FIG. 3 shows the results of sequencing the phage clone M3A4-12 capable of binding to M3-DPPE. FIG. 4 shows the results of sequencing the phage clone M3G4-10 capable of binding to M3-DPPE.

Claims (16)

A method for identifying an antibody that specifically binds to a sugar chain, comprising bringing a phage-displayed antibody into contact with the sugar chain. The method according to claim 1, wherein the sugar chain is immobilized on a solid phase. The method according to claim 1 or 2, wherein the sugar chain is a glycolipid. The method according to any one of claims 1 to 3, wherein the sugar chain is an artificial glycolipid. The sugar component constituting the sugar chain is D-mannose (D-Man), L-fucose (L-Fuc), DN-acetylglucosamine (D-GlcNAc), D-glucose (D-Glc), D- The method according to any one of claims 1 to 4, which is galactose (D-Gal), DN-acetylgalactosamine (D-GalNAc), or sialic acid. The method according to any one of claims 1 to 5, wherein the phage-displaying antibody is a human-type single-chain phage library. A phage-displayed antibody uses a human cDNA library as a template to amplify a fragment containing CDR1 and CDR2 regions of a VH or VL region of an immunoglobulin gene and a fragment containing a CDR3 region by the PCR method, mix them, and template As described above, the VH or VL region was amplified by the PCR method, and these amplified DNA fragments were each incorporated into a non-expression type phagemid vector to prepare a VH library and a VL library. These E. coli VH library and VL library Infecting a helper phage, converting it into a phage library, co-infecting these phage-type VH and VL libraries with Escherichia coli expressing Cre recombinase, and performing a recombination reaction between VH and VL vectors in E. coli. Infect this E. coli with helper phage Is obtained by making a phage expressing single chain full-length antibody, the method according to any one of claims 1 to 6. The phage-displayed antibody is recovered from a human-type single-chain phage library using the binding ability to an artificial glycolipid having a predetermined sugar chain structure as an index. the method of. A human-type single-stranded phage library was prepared, and a human-type single-stranded phage that binds to the sugar chain structure was obtained by panning using the binding ability to an artificial glycolipid having a predetermined sugar chain structure as an index. The method according to any one of claims 1 to 8, comprising contacting a human single-stranded phage with a sugar chain immobilized on a solid phase. The method according to any one of claims 1 to 9, wherein the binding between the sugar chain and the phage-displayed antibody is detected using an anti-phage antibody labeled with a detectable substance. The method according to any one of claims 1 to 10, wherein the binding between the sugar chain and the phage-displayed antibody is detected by ELISA. An antibody or antibody library that specifically binds to a specific sugar chain, which is isolated by the method according to any one of claims 1 to 11. An antibody having the amino acid sequence set forth in SEQ ID NOs: 1 and 2, which has a specific binding ability to M3-DPPE. An antibody having the amino acid sequence of SEQ ID NOs: 3 and 4, which has a specific binding ability to M3-DPPE. A DNA having a base sequence encoding the amino acid sequence set forth in SEQ ID NOs: 1 and 2. A DNA having a base sequence encoding the amino acid sequence set forth in SEQ ID NOs: 3 and 4.

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