WO2024070874A1 - モノクローナル抗体、及びａ１ベータカゼインの検出方法 - Google Patents

モノクローナル抗体、及びａ１ベータカゼインの検出方法 Download PDF

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Publication number
WO2024070874A1
WO2024070874A1 PCT/JP2023/034219 JP2023034219W WO2024070874A1 WO 2024070874 A1 WO2024070874 A1 WO 2024070874A1 JP 2023034219 W JP2023034219 W JP 2023034219W WO 2024070874 A1 WO2024070874 A1 WO 2024070874A1
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xaa
seq
thr
ser
tyr
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English (en)
French (fr)
Japanese (ja)
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誠 松山
朋絵 小林
高志 庫本
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SOWAKAI MEDICAL FOUNDATION
Tokyo University of Agriculture
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SOWAKAI MEDICAL FOUNDATION
Tokyo University of Agriculture
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Priority to JP2024549277A priority Critical patent/JP7760134B2/ja
Priority to EP23869303.0A priority patent/EP4596578A1/en
Priority to AU2023353616A priority patent/AU2023353616A1/en
Publication of WO2024070874A1 publication Critical patent/WO2024070874A1/ja
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • the present invention relates to a monoclonal antibody that binds to A1 beta-casein or to both A1 beta-casein and A2 beta-casein, and a method for detecting A1 beta-casein using the monoclonal antibody.
  • Milk contains about 3.3% protein, and about 80% of the protein in milk is casein, a phosphoprotein in which phosphate is bound to the side chain hydroxyl group of serine residues.
  • Casein is classified into alpha, beta and kappa types, and beta casein is further classified into A1 and A2 types.
  • beta caseins A1 beta casein is known to be the cause of gas generation, bloating, diarrhea and stomach discomfort when consuming milk. Therefore, although it is not yet fully recognized in Japan, the market for A2 milk, which has reduced harmful effects caused by A1 beta casein, has been expanding in Europe and the United States in recent years. Therefore, it is necessary to measure the content of A1 beta casein in milk.
  • A1 beta casein and A2 beta casein are amino acid residue at position 67 and proline
  • the ratio of A1 beta casein to A2 beta casein in milk depends on the difference in the cow's beta casein gene. In other words, cows can be divided into those that produce only A1 beta casein, those that produce only A2 beta casein, and those that produce both A1 and A2 beta caseins.
  • Patent Document 1 describes a method for determining the amount of A1 beta-casein or A2 beta-casein in a composition by liquid chromatography-mass spectrometry (LC-MS).
  • LC-MS liquid chromatography-mass spectrometry
  • the LC-MS method makes it possible to test milk products produced from multiple cows.
  • the LC-MS method requires specialized and expensive equipment, and is time-consuming and laborious due to pre-processing, etc.
  • the ELISA method which uses a specific antibody against the target substance in combination with an enzyme reaction, is known as a method for quantifying a target substance.
  • the ELISA method allows the testing of milk products instead of individual bovine samples, and is simple and does not take much time.
  • ELISA kits for quantifying A1 beta-casein are commercially available (Non-Patent Documents 1 and 2).
  • Bovine A1 Beta Casein ELISA Kit Catalog No: BEK-2364-1P Biosensis (registered trademark) Bovine A1 ⁇ -Casein ELISA Kit, Catalogue Number: BEK-2243-1P/2P/5P/10P
  • an object of the present invention is to provide a monoclonal antibody that binds to A1 beta-casein or both A1 beta-casein and A2 beta-casein, and a method for detecting A1 beta-casein using the monoclonal antibody.
  • the present inventors have conducted extensive research to solve the above problems, and as a result have developed a monoclonal antibody that specifically binds to A1 beta-casein or both A1 beta-casein and A2 beta-casein, thereby completing the present invention.
  • the present invention will now be described.
  • a heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 301, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 302, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 303; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 304, a light chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 305, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 306;
  • a monoclonal antibody characterized by binding to A1 beta-casein.
  • a heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 301, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 302, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 307; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 308, a light chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 309, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 310;
  • a monoclonal antibody characterized by binding to A1 beta-casein.
  • a heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 301, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 302, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 311; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 312, a light chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 313, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 314;
  • a monoclonal antibody characterized by binding to A1 beta-casein and A2 beta-casein.
  • a hybridoma comprising a nucleic acid encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody according to any one of [1] to [3].
  • a method for detecting A1 beta-casein in a milk sample comprising the steps of: A method comprising the step of mixing the milk sample with the monoclonal antibody described in [1] or [2]. [7] Further, a step of selecting a detection antibody from the monoclonal antibodies according to [1] or [2], and selecting a capture antibody having an epitope different from that of the detection antibody from the monoclonal antibodies according to any one of [1] to [3]; immobilizing the capture antibody on a plate; and The method according to claim 6, further comprising the step of mixing the detection antibody with the mixture of the capture antibody and the milk sample.
  • the monoclonal antibody of the present invention can specifically bind to A1 beta-casein, or both A1 beta-casein and A2 beta-casein. Therefore, the A1 beta-casein contained in a milk sample can be easily detected or quantified by an ELISA method using the monoclonal antibody of the present invention. Therefore, the present invention is extremely advantageous from an industrial perspective as a technology that can easily evaluate milk by focusing on A1 beta-casein, which has been attracting attention in recent years.
  • FIG. 1 is a graph showing the results of quantifying A1 beta-casein in milk samples by sandwich ELISA using the anti-A1 beta-casein antibody and anti-A1/A2 beta-casein antibody of the present invention.
  • FIG. 2 is a graph showing the results of quantifying A1 beta-casein in milk samples by the sandwich ELISA method using the anti-A1 beta-casein antibody of the present invention.
  • FIG. 3 is a graph showing the results of quantifying A1 beta-casein in milk samples by the sandwich ELISA method using the anti-A1 beta-casein antibody of the present invention.
  • FIG. 1 is a graph showing the results of quantifying A1 beta-casein in milk samples by sandwich ELISA using the anti-A1 beta-casein antibody and anti-A1/A2 beta-casein antibody of the present invention.
  • FIG. 2 is a graph showing the results of quantifying A1 beta-casein in milk samples by the sandwich ELISA method using the anti-A1 beta-casein antibody of the present invention.
  • FIG. 4 is a graph showing the results of quantifying A1 beta-casein in milk samples by sandwich ELISA using the anti-A1 beta-casein antibody and anti-A1/A2 beta-casein antibody of the present invention.
  • FIG. 5 is a graph showing the results of quantifying A1 beta-casein in milk samples by sandwich ELISA using the anti-A1 beta-casein antibody and anti-A1/A2 beta-casein antibody of the present invention.
  • the anti-A1 beta casein monoclonal antibody obtained by the inventors from rat cells may be referred to as "monoclonal antibody (I)", the anti-A1 beta casein monoclonal antibody obtained from mouse cells as “monoclonal antibody (II)”, and the anti-A1/A2 beta casein monoclonal antibody obtained from rat cells as “monoclonal antibody (III)".
  • the anti-A1/A2 beta casein monoclonal antibody is an antibody whose antigens are both A1 beta casein and A2 beta casein. In the following sequence definitions, "/" indicates “or”, and “deletion” indicates that the amino acid residue may be absent.
  • Monoclonal antibody (I) comprises a heavy chain variable region comprising heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 301, heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of SEQ ID NO: 302, and heavy chain complementarity determining region 3 (VH CDR3) comprising the amino acid sequence of SEQ ID NO: 303; and a light chain variable region comprising light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO: 304, light chain complementarity determining region 2 (VL CDR2) comprising the amino acid sequence of SEQ ID NO: 305, and light chain complementarity determining region 3 (VL CDR3) comprising the amino acid sequence of SEQ ID NO: 306; and binds to A1 beta casein.
  • VH CDR1 heavy chain complementarity determining region 1
  • VH CDR2 heavy chain complementarity determining region 2
  • Xaa1 - Xaa2- Xaa3 - Xaa4 - Xaa5 - Xaa6 - Xaa7 - Xaa8 Xaa 1 Gly/Ala
  • Xaa 2 Phe/Tyr/Trp/Val/Leu/Ile/Thr/Ser
  • Xaa 3 Thr/Ser/Val/Leu/Ile/Pro/Asp/Glu
  • Xaa 4 Phe/Tyr/Trp/Val/Leu/Ile
  • Xaa 5 Ser/Thr/Arg/Lys/His/Asn/Gln/Asp/Glu
  • Xaa 6 Asn/Gln/Ser/Thr/Asp/Glu/Arg/Lys/His
  • Xaa 7 Tyr/Phe/Trp/Thr/Ser/Lys/Arg/His/Asn/G
  • SEQ ID NO: 301 includes the following SEQ ID NO: 501.
  • Xaa 701 Gly/Ala
  • Xaa 702 Phe/Tyr/Trp
  • Xaa 703 Thr/Ser
  • Xaa 704 Phe/Tyr/Trp
  • Xaa 705 Ser/Thr/Arg/Lys/His
  • Xaa 706 Asn/Gln/Ser/Thr
  • Xaa 707 Tyr/Phe/Trp
  • Xaa 708 Asp/Glu/Gly/Ala/Thr/Ser
  • SEQ ID NO:301 the following SEQ ID NOs:336 to 344 are preferred.
  • Xaa 299 Gly/Ala
  • Xaa 300 Tyr/Phe/Trp
  • Xaa 301 Thr/Ser
  • Xaa 302 Tyr/Phe/Trp
  • Xaa 303 Thr/Ser
  • Xaa 304 Asn/Gln
  • Xaa 305 Tyr/Phe/Trp
  • Xaa 306 Asp/Glu
  • Xaa 307 Gly/Ala
  • Xaa 308 Tyr/Phe/Trp
  • Xaa 309 Thr/Ser
  • Xaa 310 Tyr/Phe/Trp
  • Xaa 311 Thr/Ser
  • Xaa 312 Asn/Gln
  • Xaa 313 Tyr/Phe/Trp
  • Xaa 314 Gly/Ala
  • Xaa 315 Gly/Ala
  • Xaa 316 Tyr/Phe/Trp
  • Xaa 317 Thr/Ser
  • Xaa 318 Tyr/Phe/Trp
  • Xaa 319 Thr/Ser
  • Xaa 320 Thr/Ser
  • Xaa 321 Tyr/Phe/Trp
  • Xaa 322 Gly/Ala
  • Xaa323 Gly/Ala
  • Xaa324 Tyr/Phe/Trp
  • Xaa325 Thr/Ser
  • Xaa326 Tyr/Phe/Trp
  • Xaa327 Lys/Arg/His
  • Xaa328 Asn/Gln
  • Xaa329 Tyr/Phe/Trp
  • Xaa330 Asp/Glu
  • Xaa 339 Gly/Ala
  • Xaa 340 Tyr/Phe/Trp
  • Xaa 341 Val/Leu/Ile
  • Xaa 342 Tyr/Phe/Trp
  • Xaa 343 Arg/Lys/His
  • Xaa 344 Asn/Gln
  • Xaa 345 Tyr/Phe/Trp
  • Xaa 346 Gly/Ala
  • SEQ ID NO: 342 Xaa 347 -Xaa 348 -Xaa 349 -Xaa 350 -Xaa 351 -Xaa 352 -Xaa 353 -Xaa 354
  • Xaa 347 Gly/Ala
  • Xaa 348 Tyr/Phe/Trp
  • Xaa 349 Thr/Ser
  • Xaa 350 Tyr/Phe/Trp
  • Xaa 351 Thr/Ser
  • Xaa 352 Asn/Gln
  • Xaa 353 Tyr/Phe/Trp
  • Xaa 354 Tyr/Phe/Trp
  • Xaa 355 Gly/Ala
  • Xaa 356 Tyr/Phe/Trp
  • Xaa 357 Thr/Ser
  • Xaa 358 Tyr/Phe/Trp
  • Xaa 359 Thr/Ser
  • Xaa 360 Asn/Gln
  • Xaa 361 Tyr/Phe/Trp
  • Xaa 362 Gly/Ala
  • SEQ ID NO: 344 Xaa 363 -Xaa 364 -Xaa 365 -Xaa 366 -Xaa 367 -Xaa 368 -Xaa 369 -Xaa 370
  • Xaa 363 Gly/Ala
  • Xaa 364 Tyr/Phe/Trp
  • Xaa 365 Thr/Ser
  • Xaa 366 Tyr/Phe/Trp
  • Xaa 367 Asp/Glu
  • Xaa 368 Asp/Glu
  • Xaa 369 Tyr/Phe/Trp
  • Xaa 370 Gly/Ala
  • Xaa9 Ile/Val/Leu/Phe/Tyr/Trp
  • Xaa10 Thr/Ser/Asn/Gln/Lys/Arg/His/Gly/Ala/Tyr/Phe/Trp
  • Xaa11 Thr/Ser/Pro/Tyr/Phe/Trp/Gly/Ala/Asn/Gln/Val/Leu/Ile/Asp/Glu
  • Xaa12 Ser/Thr/Asp/Glu/Lys/Arg/His/Phe/Tyr/Trp/Gly/Ala/Asn/Gln
  • Xaa13 Xaa14 Gly/Ala/Ser
  • SEQ ID NO: 302 includes SEQ ID NO: 502 below.
  • Xaa 709 Ile/Val/Leu/Phe/Tyr/Trp
  • Xaa 710 Thr/Ser/Asn/Gln
  • Xaa 711 Thr/Ser/Pro/Tyr/Phe/Trp
  • Xaa 712 Ser/Thr/Asp/Glu
  • Xaa 713 Gly/Ala/Ser/Thr
  • Xaa 714 Gly/Ala/Ser/Thr
  • Xaa 715 Ser/Thr/Tyr/Phe/Trp
  • Xaa 716 Asp/Glu/Gly/Ala/Thr/Ser
  • SEQ ID NO:302 the following SEQ ID NOs:345 to 353 are preferred.
  • Xaa 371 Val/Leu/Ile
  • Xaa 372 Thr/Ser
  • Xaa 373 Thr/Ser
  • Xaa 374 Thr/Ser
  • Xaa 375 Gly/Ala
  • Xaa 376 Gly/Ala
  • Xaa 377 Thr/Ser
  • Xaa 378 Thr/Ser
  • Xaa 379 Val/Leu/Ile
  • Xaa 380 Asn/Gln
  • Xaa 381 Tyr/Phe/Trp
  • Xaa 382 Asp/Glu
  • Xaa 383 Gly/Ala
  • Xaa 384 Thr/Ser
  • Xaa 385 Thr/Ser
  • Xaa 386 Thr/Ser
  • SEQ ID NO: 348 Xaa 395 -Xaa 396 -Xaa 397 -Xaa 398 -Xaa 399 -Xaa 400 -Xaa 401 -Xaa 402
  • Xaa 395 Val/Leu/Ile
  • Xaa 396 Asn/Gln
  • Xaa 397 Pro
  • Xaa 398 Thr/Ser
  • Xaa 399 Thr/Ser
  • Xaa 400 Gly/Ala
  • Xaa 401 Tyr/Phe/Trp
  • Xaa 402 Val/Leu/Ile
  • Xaa 403 Val/Leu/Ile
  • Xaa 404 Asn/Gln
  • Xaa 405 Pro
  • Xaa 406 Thr/Ser
  • Xaa 407 Thr/Ser
  • Xaa 408 Gly/Ala
  • Xaa 409 Tyr/Phe/Trp
  • Xaa 410 Thr/Ser
  • Xaa 419 Val/Leu/Ile
  • Xaa 420 Asn/Gln
  • Xaa 421 Thr/Ser
  • Xaa 422 Tyr/Phe/Trp
  • Xaa 423 Thr/Ser
  • Xaa 424 Gly/Ala
  • Xaa 425 Asp/Glu
  • Xaa 426 Thr/Ser
  • Xaa 435 Val/Leu/Ile
  • Xaa 436 Thr/Ser
  • Xaa 437 Tyr/Phe/Trp
  • Xaa 438 Gly/Ala
  • Xaa 439 Gly/Ala
  • Xaa 440 Arg/Lys/His
  • Xaa 441 Thr/Ser
  • Xaa 442 Val/Leu/Ile
  • SEQ ID NO: 303 includes SEQ ID NO: 503 below.
  • Xaa 717 Ala/Gly
  • Xaa 718 Arg/Lys/His
  • Xaa 719 His/Arg/Lys/deletion
  • Xaa 720 Pro/Gly/Ala/Ser/Thr
  • Xaa 721 Tyr/Phe/Trp
  • sequence number 303 the following sequence numbers 315, 316, and 354 to 356 are preferred.
  • Xaa 479 Ser/Thr
  • Xaa 480 His/Arg/Lys
  • Xaa 481 Asp/Glu
  • Xaa 482 His/Arg/Lys
  • Xaa 483 Gly/Ala
  • Xaa 484 His/Arg/Lys
  • Xaa 485 Tyr/Phe/Trp
  • Xaa 486 Val/Leu/Ile
  • Xaa 487 Tyr/Phe/Trp
  • Xaa 488 Asp/Glu
  • Xaa 489 Tyr/Phe/Trp
  • SEQ ID NO: 304 Xaa 38 -Xaa 39 -Xaa 40 -Xaa 41 -Xaa 42 -Xaa 43 -Xaa 44 -Xaa 45 -Xaa 46 -Xaa 47 -Xaa 48
  • Xaa 38 Gln/Asn/Ser/Thr/Lys/Arg/His
  • Xaa 39 Asn/Gln/Ser/Thr/Arg/Lys/His
  • Xaa 40 Ile/Val/Leu
  • Xaa 41 Ser/Thr/Ile/Val/Leu/Asp/Glu/deletion
  • Xaa 42 Lys/Arg/His/Tyr/Phe/Trp/deletion
  • Xaa 43 Ser/Thr/Tyr/Phe/Trp/deletion
  • Xaa 44 Ser/Thr/Asn/Gln/Asp/Glu/deletion
  • SEQ ID NO: 304 includes SEQ ID NO: 504 below.
  • Xaa 737 Gln/Asn/Ser/Thr
  • Xaa 738 Asn/Gln/Ser/Thr/Arg/Lys/His
  • Xaa 739 Ile/Val/Leu
  • Xaa 740 Ser/Thr/deletion
  • Xaa 741 Tyr/Phe/Trp
  • Xaa 742 Lys/Arg/His/deletion
  • Xaa 743 Asn/Gln/deletion
  • sequence number 304 the following sequence numbers 317, 318, 357 to 359 are preferred.
  • Xaa158 Gln/Asn
  • Xaa159 Asn/Gln
  • Xaa160 Ile/Val/Leu
  • Xaa161 Tyr/Phe/Trp
  • Xaa162 Lys/Arg/His
  • Xaa163 Asn/Gln
  • Xaa 164 Ser/Thr
  • Xaa165 Ser/Thr/Arg/Lys/His
  • Xaa166 Ile/Val/Leu
  • Xaa167 Ser/Thr
  • Xaa168 Tyr/Phe/Trp
  • Xaa 490 Ser/Thr
  • Xaa 491 His/Arg/Lys
  • Xaa 492 Ile/Val/Leu
  • Xaa 493 Ser/Thr
  • Xaa 494 Tyr/Phe/Trp
  • Xaa 495 Ser/Thr
  • Xaa 496 Ser/Thr
  • Xaa 497 Ile/Val/Leu
  • Xaa 498 Ser/Thr
  • Xaa 499 Tyr/Phe/Trp
  • Xaa 500 Asn/Gln
  • Xaa501 Asn/Gln
  • Xaa502 Ile/Val/Leu
  • Xaa503 Asn/Gln
  • Xaa504 Asn/Gln
  • Xaa505 Tyr/Phe/Trp
  • Xaa 49 Asn/Gln/Asp/Glu/Ile/Val/Leu/Thr/Ser/Tyr/Phe/Trp
  • Xaa 50 Ala/Gly/Thr/Ser/Ile/Val/Leu/Pro/Cys/Met
  • Xaa 51 Asn/Gln/Ser/Thr
  • SEQ ID NO: 305 includes the following SEQ ID NO: 505.
  • SEQ ID NO: 505 Xaa 744 -Xaa 745 -Xaa 746
  • Xaa 744 Asn/Gln/Asp/Glu
  • Xaa 745 Ala/Gly/Thr/Ser
  • Xaa 746 Asn/Gln/Ser/Thr
  • SEQ ID NO:305 the following SEQ ID NOs:319, 320 and 360 are preferred.
  • Xaa 52 Gln/Asn/Ile/Val/Leu/Tyr/Phe/Trp/deletion
  • Xaa 53 Gln/Asn/Lys/Arg/His
  • Xaa 54 Tyr/Phe/Trp/Ser/Thr/Gly/Ala/Lys/Arg/His
  • Xaa 55 Tyr/Phe/Trp/Ser/Thr/Gln/Asn/Leu/Val/Ile/Lys/Arg/His/deletion
  • Xaa 56 Ser/Thr/Lys/Arg/His/Asp/Glu/deletion
  • SEQ ID NO: 306 includes SEQ ID NO: 506 below.
  • Xaa 747 Gln/Asn
  • Xaa 748 Gln/Asn
  • Xaa 749 Tyr/Phe/Trp
  • Xaa 750 Tyr/Phe/Trp/deletion
  • Xaa 751 Ser/Thr
  • Xaa 752 Gln/Asn/Ser/Thr/deletion
  • Xaa 753 Thr/Ser/deletion
  • Xaa 754 Gly/Ala/Pro
  • Xaa 755 Leu/Val/Ile/Tyr/Phe/Trp
  • SEQ ID NO:306 the following SEQ ID NOs:321, 322, 361 to 363 are preferred.
  • Xaa175 Gln/Asn
  • Xaa176 Gln/Asn
  • Xaa177 Tyr/Phe/Trp
  • Xaa178 Tyr/Phe/Trp
  • Xaa179 Ser/Thr
  • Xaa180 Gly/Ala/Pro
  • Xaa181 Leu/Val/Ile
  • Xaa182 Thr/Ser
  • Xaa 509 Gln/Asn
  • Xaa 510 Gln/Asn
  • Xaa 511 Tyr/Phe/Trp
  • Xaa 512 Ser/Thr
  • Xaa 513 Ser/Thr
  • Xaa 514 Ser/Thr
  • Xaa 515 Pro
  • Xaa 516 Tyr/Phe/Trp
  • Xaa 517 Ser/Thr
  • Xaa 518 Gln/Asn
  • Xaa 519 Gln/Asn
  • Xaa 520 Tyr/Phe/Trp
  • Xaa 521 Ser/Thr
  • Xaa 522 Gln/Asn
  • Xaa 523 Ser/Thr
  • Xaa 524 Pro
  • Xaa 525 Tyr/Phe/Trp
  • Xaa 526 Ser/Thr
  • Monoclonal antibody (II) comprises a heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 301, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 302, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 307; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 308, a light chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 309, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 310; and binds to A1 beta-casein.
  • SEQ ID NO: 307 Xaa 62 -Xaa 63 -Xaa 64 -Xaa 65 -Xaa 66 -Xaa 67 -Xaa 68 -Xaa 69 -Xaa 70 -Xaa 71 -Xaa 72 -Xaa 73 -Xaa 74 -Xaa 75 -Xaa 76 -Xaa 77 -Xaa 78
  • Xaa 62 Ala/Gly/Ser/Thr/Val/Leu/Ile
  • Xaa 63 Val/Leu/Ile/Gly/Ala/Lys/Arg/His/deletion
  • Xaa 64 Tyr/Phe/Trp/Pro/Asp/Glu/Lys/Arg/His/deletion
  • Xaa 65 Asp/Glu/Ala/Gly/Ser/Thr/Lys/Arg/His/Pro/deletion
  • SEQ ID NO: 307 includes the following SEQ ID NO: 507.
  • SEQ ID NO:507 Xaa 757 -Xaa 758 -Xaa 759 -Xaa 760 -Xaa 761 -Xaa 762 -Xaa 763 -Xaa 764 -Xaa 765 -Xaa 766 -Xaa 767 -Xaa 768
  • Xaa 757 Ala/Gly
  • Xaa 758 Val/Leu/Ile/Gly/Ala
  • Xaa 759 Tyr/Phe/Trp
  • Xaa 760 Asp/Glu/Ala/Gly
  • Xaa 761 Gly/Ala/Lys/Arg/His
  • Xaa 762 Tyr/Phe/Trp/Asp/Glu
  • Xaa 763 Tyr/Phe/Trp
  • Xaa 764 Pro/Val/Leu/Ile
  • SEQ ID NO:307 the following SEQ ID NOs:323, 324, 364 to 366 are preferred.
  • SEQ ID NO: 324 Xaa 203 -Xaa 204 -Xaa 205 -Xaa 206 -Xaa 207 -Xaa 208 -Xaa 209 -Xaa 210 -Xaa 211 -Xaa 212 -Xaa 213
  • Xaa 203 Ala/Gly
  • Xaa 204 Gly/Ala
  • Xaa 205 Tyr/Phe/Trp
  • Xaa 206 Ala/Gly
  • Xaa 207 Lys/Arg/His
  • Xaa 208 Asp/Glu
  • Xaa 209 Tyr/Phe/Trp
  • Xaa 210 Pro/Val/Leu/Ile
  • Xaa 211 Met/Cys
  • Xaa 212 Asp/Glu
  • Xaa 213 Tyr/Phe/Trp
  • Xaa 535 Gly/Ala
  • Xaa 536 Lys/Arg/His
  • Xaa 537 Gln/Asn
  • Xaa 538 Tyr/Phe/Trp
  • Xaa 539 Tyr/Phe/Trp
  • Xaa 540 Gly/Ala
  • Xaa 541 Val/Leu/Ile
  • Xaa 542 Ser/Thr
  • Xaa 543 Gly/Ala
  • Xaa 544 Asp/Glu
  • Xaa 545 Tyr/Phe/Trp
  • SEQ ID NO: 365 Xaa 546 -Xaa 547 -Xaa 548 -Xaa 549 -Xaa 550 -Xaa 551 -Xaa 552 -Xaa 553 -Xaa 554 -Xaa 555 -Xaa 556 -Xaa 557 -Xaa 558 -Xaa 559 -Xaa 560 -Xaa 561
  • Xaa 546 Gly/Ala
  • Xaa 547 Lys/Arg/His
  • Xaa 548 Lys/Arg/His
  • Xaa 549 Gly/Ala
  • Xaa 550 Val/Leu/Ile
  • Xaa 551 Tyr/Phe/Trp
  • Xaa 552 Gln/Asn
  • Xaa 553 Tyr/Phe/Trp
  • Xaa 554 Asp/Glu
  • SEQ ID NO: 366 Xaa 562 -Xaa 563 -Xaa 564 -Xaa 565 -Xaa 566 -Xaa 567 -Xaa 568 -Xaa 569 -Xaa 570 -Xaa 571 -Xaa 572 -Xaa 573 -Xaa 574 -Xaa 575 -Xaa 576 -Xaa 577 -Xaa 578
  • Xaa 562 Gly/Ala
  • Xaa 563 Lys/Arg/His
  • Xaa 564 Lys/Arg/His
  • Xaa 565 Gly/Ala
  • Xaa 566 Tyr/Phe/Trp
  • Xaa 567 Lys/Arg/His
  • Xaa 568 Tyr/Phe/Trp
  • Xaa 569 Ser/Thr
  • Xaa 570 Gln/Asn
  • Xaa 79 Gln/Asn/Ser/Thr/Lys/Arg/His
  • Xaa 80 Ser/Thr/Gly/Ala
  • Xaa 81 Leu/Val/Ile
  • Xaa 82 Leu/Val/Ile/Tyr/Phe/Trp/Ser/Thr/deletion
  • Xaa 83 Asn/Gln/Arg/Lys/His/Ser/Thr/Asp/Glu/deletion
  • Xaa 84 Ser/Thr/Leu/Val/Ile/deletion
  • Xaa 85 Arg/Ly
  • SEQ ID NO:308 includes the following SEQ ID NO:508.
  • Xaa 768 Gln/Asn
  • Xaa 769 Ser/Thr
  • Xaa 770 Leu/Val/Ile
  • Xaa 771 Leu/Val/Ile
  • Xaa 772 Asn/Gln
  • Xaa 773 Ser/Thr
  • Xaa 774 Arg/Lys/His
  • Xaa 775 Thr/Ser
  • Xaa 776 Arg/Lys/His
  • Xaa 777 Lys/Arg/His,
  • SEQ ID NO:308 the following SEQ ID NOs:367 to 371 are preferred.
  • Xaa 579 Gln/Asn
  • Xaa 580 Ser/Thr
  • Xaa 581 Val/Leu/Ile
  • Xaa 582 Val/Leu/Ile
  • Xaa 583 Gln/Asn
  • Xaa 584 Ser/Thr
  • Xaa 585 Arg/Lys/His
  • Xaa 586 Ser/Thr
  • Xaa 587 Arg/Lys/His
  • Xaa 588 Arg/L
  • SEQ ID NO: 368 Xaa 591 -Xaa 592 -Xaa 593 -Xaa 594 -Xaa 595 -Xaa 596 -Xaa 597 -Xaa 598 -Xaa 599 -Xaa 600 -Xaa 601
  • Xaa 591 Gln/Asn
  • Xaa 592 Gly/Ala
  • Xaa 593 Val/Leu/Ile
  • Xaa 594 Val/Leu/Ile
  • Xaa 595 Arg/Lys/His
  • Xaa 596 Ser/Thr
  • Xaa 597 Gln/Asn
  • Xaa 598 Gly/Ala
  • Xaa 599 Gln/Asn
  • Xaa 600 Ser/Thr
  • Xaa 601 Tyr/Phe/Trp
  • Xaa 602 Arg/Lys/His
  • Xaa 603 Ser/Thr
  • Xaa 604 Val/Leu/Ile
  • Xaa 605 Ser/Thr
  • Xaa 606 Ser/Thr
  • Xaa 607 Ser/Thr
  • Xaa 608 Gly/Ala
  • Xaa 609 Tyr/Phe/Trp
  • Xaa 610 Ser/Thr
  • Xaa 611 Tyr/Phe/Trp
  • Xaa 612 Asn/Gln
  • Xaa 613 Ser/Thr
  • Xaa 614 Val/Leu/Ile
  • Xaa 615 Val/Leu/Ile
  • Xaa 616 Arg/Lys/His
  • Xaa 617 Ser/Thr
  • Xaa 618 Asn/Gln
  • Xaa 619 Gly/Ala
  • Xaa 620 Asn/Gln
  • Xaa 621 Ser/Thr
  • Xaa 622 Tyr/Phe/Trp
  • SEQ ID NO: 309 includes SEQ ID NO: 509 below.
  • SEQ ID NO:309 the following SEQ ID NOs:372 to 374 are preferred.
  • Xaa 94 Gln/Asn/Lys/Arg/His/Tyr/Phe/Trp/Ser/Thr
  • Xaa 95 Gln/Asn/Lys/Arg/His
  • Xaa 96 Ser/Thr/Asp/Glu/Ala/Gly/Tyr/Phe/Trp/Val/Leu/Ile
  • Xaa 97 Tyr/Phe/Trp/Ser/Thr/Ala/Gly/deletion
  • Xaa 98 Asn/Gln/Thr/Ser/Lys/Arg/His/Leu/Val/Ile
  • Xaa 99 Leu/Val/Ile/Tyr
  • SEQ ID NO: 310 includes SEQ ID NO: 510 below.
  • Xaa 783 Gln/Asn/Lys/Arg/His
  • Xaa 784 Gln/Asn
  • Xaa 785 Ser/Thr
  • Xaa 786 Tyr/Phe/Trp
  • Xaa 787 Asn/Gln/Thr/Ser
  • Xaa 788 Leu/Val/Ile
  • Xaa 789 Pro/Val/Leu/Ile
  • Xaa 790 Trp/Phe/Tyr
  • Xaa 791 Thr/Ser
  • SEQ ID NO:310 the following SEQ ID NOs:325, 326, 375 and 376 are preferred.
  • SEQ ID NO: 325 Xaa 214 -Xaa 215 -Xaa 216 -Xaa 217 -Xaa 218 -Xaa 219 -Xaa 220 -Xaa 221 -Xaa 222
  • Xaa 214 Gln/Asn
  • Xaa 215 Gln/Asn
  • Xaa 216 Ser/Thr
  • Xaa 217 Tyr/Phe/Trp
  • Xaa 218 Asn/Gln
  • Xaa 219 Leu/Val/Ile
  • Xaa 220 Pro/Val/Leu/Ile
  • Xaa 221 Trp/Phe/Tyr
  • Xaa 222 Thr/Ser
  • SEQ ID NO: 326 Xaa 223 -Xaa 224 -Xaa 225 -Xaa 226 -Xaa 227 -Xaa 228 -Xaa 229 -Xaa 230 -Xaa 231
  • Xaa 223 Lys/Arg/His
  • Xaa 224 Gln/Asn
  • Xaa 225 Ser/Thr
  • Xaa 226 Tyr/Phe/Trp
  • Xaa 227 Thr/Ser
  • Xaa 228 Leu/Val/Ile
  • Xaa 229 Pro/Val/Leu/Ile
  • Xaa 230 Trp/Phe/Tyr
  • Xaa 231 Thr/Ser
  • Xaa 643 Ser/Thr/His
  • Xaa 644 Asn/Gln
  • Xaa 645 Ser/Thr
  • Xaa 646 Ser/Thr
  • Xaa 647 Lys/Arg/His
  • Xaa 648 Val/Leu/Ile
  • Xaa 649 Pro
  • Xaa 650 Tyr/Phe/Trp
  • Xaa 651 Ser/Thr
  • Xaa 652 Asn/Gln
  • Xaa 653 Lys/Arg/His
  • Xaa 654 Val/Leu/Ile
  • Xaa 655 Lys/Arg/His
  • Xaa 656 Asp/Glu
  • Xaa 657 Val/Leu/Ile
  • Xaa 658 Ser/Thr
  • Xaa 659 Lys/Arg/His
  • the monoclonal antibody (III) comprises a heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO:301, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO:302, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO:311; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO:312, a light chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO:313, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO:314; and binds to A1 beta-casein and A2 beta-casein.
  • Xaa 102 Val/Leu/Ile/Ala/Gly/Ser/Thr
  • Xaa 103 Arg/Lys/His/Ser/Thr
  • Xaa 104 His/Lys/Arg/Gly/Ala/Val/Leu/Ile/deletion
  • Xaa 105 Gly/Ala/Ser/Thr/deletion
  • Xaa 106 Arg/His/Lys/Gly/Ala/deletion
  • Xaa 107 Arg/His/Lys/Ser/Thr/Phe/Ty
  • SEQ ID NO:311 includes SEQ ID NO:511 below.
  • Xaa 792 Val/Leu/Ile/Ala/Gly
  • Xaa 793 Arg/Lys/His
  • Xaa 794 His/Lys/Arg
  • Xaa 795 Gly/Ala/deletion
  • Xaa 796 Arg/His/Lys
  • Xaa 797 Arg/His/Lys/Ser/Thr
  • Xaa 798 Gln/Asn/deletion
  • Xaa 799 Phe/Tyr/Trp
  • Xaa 800 Asp/Glu/Pro/
  • SEQ ID NO:311 the following SEQ ID NOs:327 to 329 and 377 are preferred.
  • Xaa 232 Val/Leu/Ile
  • Xaa 233 Arg/Lys/His
  • Xaa 234 His/Lys/Arg
  • Xaa 235 Gly/Ala
  • Xaa 236 Arg/His/Lys
  • Xaa 237 Arg/His/Lys
  • Xaa 238 Phe/Tyr/Trp
  • Xaa 239 Asp/Glu
  • Xaa 240 Phe/Tyr/Trp
  • Xaa 241 Ala/Gly
  • Xaa 242 Arg/Lys/His
  • Xaa 243 His/Lys/Arg
  • Xaa 244 Arg/His/Lys
  • Xaa 245 Ser/Thr
  • Xaa 246 Phe/Tyr/Trp
  • Xaa 247 Pro/Ala/Gly
  • Xaa 248 Phe/Tyr/Trp
  • Xaa 249 Ala/Gly
  • Xaa 250 Arg/Lys/His
  • Xaa 251 His/Lys/Arg
  • Xaa 252 Gly/Ala
  • Xaa 253 Arg/His/Lys
  • Xaa 254 Ser/Thr
  • Xaa 255 Gln/Asn
  • Xaa 256 Phe/Tyr/Trp
  • Xaa 257 Ala/Gly
  • Xaa 258 Phe/Tyr/Trp
  • SEQ ID NO:312 includes SEQ ID NO:512 below.
  • Xaa 802 Gln/Asn
  • Xaa 803 Asn/Gln/deletion
  • Xaa 804 Ser/Thr/deletion
  • Xaa 805 Ile/Val/Leu
  • Xaa 806 Val/Leu/Ile/deletion
  • Xaa 807 His/Lys/Arg/deletion
  • Xaa 808 Ser/Thr/deletion
  • Xaa 809 Asp/Glu/deletion
  • Xaa 810 Gly/Ala/d
  • SEQ ID NO:312 the following SEQ ID NOs:330, 331 and 378 are preferred.
  • Xaa 258 Gln/Asn
  • Xaa 259 Asn/Gln
  • Xaa 260 Ile/Val/Leu
  • Xaa 261 Asn/Gln
  • Xaa 262 Lys/Arg/His
  • Xaa 263 Tyr/Phe/Trp
  • Xaa 264 Gln/Asn
  • Xaa 265 Ser/Thr
  • Xaa 266 Ile/Val/Leu
  • Xaa 267 Val/Leu/Ile
  • Xaa 268 His/Lys/Arg
  • Xaa 269 Ser/Thr
  • Xaa 270 Asp/Glu
  • Xaa 271 Gly/Ala
  • Xaa 272 Asn/Gln
  • Xaa 273 Thr/Ser
  • Xaa 274 Tyr/Phe/Trp
  • SEQ ID NO: 313 includes the following SEQ ID NO: 513.
  • Xaa 814 Asn/Gln/Arg/Lys/His
  • Xaa 815 Thr/Ser/Val/Leu/Ile
  • Xaa 816 Asn/Gln/Ser/Thr
  • SEQ ID NO:313 the following SEQ ID NOs:332, 333 and 379 are preferred.
  • Xaa 278 Arg/Lys/His
  • Xaa 279 Val/Leu/Ile
  • Xaa 280 Ser/Thr
  • Xaa 130 Cys/Met/Phe/Tyr/Trp/Leu/Val/Ile
  • Xaa131 Gln/Asn
  • Xaa132 His/Lys/Arg/Ser/Thr/Ala/Gly
  • Xaa133 Asn/Gln/Ala/Gly/Asp/Glu/His/Lys/Arg/Ser/Thr
  • Xaa134 Ser/Thr/His/Lys/Arg/Asn/Gln
  • Xaa135 Trp/Phe/Tyr/Ala/Gly
  • Xaa136 Pro/Val/Leu/Ile
  • Xaa137 Val/Leu/I
  • SEQ ID NO:314 includes the following SEQ ID NO:514.
  • SEQ ID NO:514 Xaa 817 -Xaa 818 -Xaa 819 -Xaa 820 -Xaa 821 -Xaa 822 -Xaa 823 -Xaa 824 -Xaa 825
  • Xaa 817 Cys/Met/Phe/Tyr/Trp/Leu/Val/Ile
  • Xaa 818 Gln/Asn
  • Xaa 819 His/Lys/Arg/Ser/Thr
  • Xaa 820 Asn/Gln/Ala/Gly
  • Xaa 821 Ser/Thr/His/Lys/Arg
  • Xaa 822 Trp/Phe/Tyr
  • Xaa 823 Pro/Val/Leu/Ile
  • Xaa 824 Val/Leu/Ile
  • Xaa 825 Thr/
  • SEQ ID NO:3134 Xaa 281 -Xaa 282 -Xaa 283 -Xaa 284 -Xaa 285 -Xaa 286 -Xaa 287 -Xaa 288 -Xaa 289
  • Xaa 281 Cys/Met/Phe/Tyr/Trp
  • Xaa 282 Gln/Asn
  • Xaa 283 His/Lys/Arg
  • Xaa 284 Asn/Gln
  • Xaa 285 Ser/Thr
  • Xaa 286 Trp/Phe/Tyr
  • Xaa 287 Pro/Val/Leu/Ile
  • Xaa 288 Val/Leu/Ile
  • Xaa 289 Thr/Ser
  • Xaa 290 Leu/Val/Ile
  • Xaa 291 Gln/Asn
  • Xaa 292 Ser/Thr
  • Xaa 293 Ala/Gly
  • Xaa 294 His/Lys/Arg
  • Xaa 295 Trp/Phe/Tyr
  • Xaa 296 Pro/Val/Leu/Ile
  • Xaa 297 Val/Leu/Ile
  • Xaa 298 Thr/Ser
  • Xaa 682 Val/Leu/Ile
  • Xaa 683 Gln/Asn
  • Xaa 684 His/Lys/Arg
  • Xaa 685 His/Lys/Arg
  • Xaa 686 Gln/Asn
  • Xaa 687 Trp/Phe/Tyr
  • Xaa 688 Pro
  • Xaa 689 Pro
  • Xaa 690 Thr/Ser
  • Antibodies are glycoproteins produced by lymphocyte B cells, and have the function of recognizing and binding to specific proteins and other molecules. Antibodies have the function of specifically binding to specific molecules called antigens, and the function of detoxifying and removing factors that have antigens in cooperation with other biological molecules and cells. Antibodies basically have the same molecular structure, and have a four-chain "Y" shape consisting of two light chains and two heavy chain polypeptides. There are two types of light chains (L chains), lambda chains and kappa chains, and all antibodies have one of these.
  • H chains There are five types of heavy chains (H chains), which have different structures: gamma chains, ⁇ chains, alpha chains, delta chains, and epsilon chains, and the type of antibody (isotype) changes depending on the difference in the heavy chain.
  • Antibodies are monomeric immunoglobulins, consisting of two heavy chains (gamma chains) and two light chains, and have two antigen-binding sites.
  • the Fc region has an effector function that triggers a reaction after the antibody binds to an antigen, and the Fab region has the function of binding to the antigen.
  • the Fab region and Fc region of the heavy chain are connected by a hinge, and the proteolytic enzyme papain contained in papaya breaks down this hinge into two Fab regions and one Fc region.
  • the domain of the Fab region near the tip of the "Y” is called the variable region (V region) because there are various changes in the amino acid sequence so that it can bind to various antigens.
  • variable region of the light chain is called the VL region
  • variable region of the heavy chain is called the VH region
  • the Fab region and Fc region other than the V region are relatively constant regions and are called constant regions (C regions).
  • the constant region of the light chain is called the CL region
  • the constant region of the heavy chain is called the CH region, which is further divided into three regions, CH1 to CH3.
  • the Fab region of the heavy chain consists of the VH region and CH1, and the Fc region of the heavy chain consists of CH2 and CH3.
  • the hinge region is located between CH1 and CH2.
  • the heavy chain and light chain each contain a variable region consisting of about 100 or more amino acids, for example 130 to 140 amino acids, that are mainly involved in antigen binding.
  • the variable region of the heavy chain is also referred to as the heavy chain variable region (VH).
  • the variable region of the light chain is also referred to as the light chain variable region (VL).
  • Each variable region contains a complementarity determining region (CDR) that forms an antigen binding site.
  • Each of the VH and VL contains three CDRs. That is, the VH contains a first CDR (HCDR1), a second CDR (HCDR2), and a third CDR (HCDR3).
  • the VL contains a first CDR (LCDR1), a second CDR (LCDR2), and a third CDR (LCDR3).
  • the regions other than the CDRs of the heavy chain and light chain have amino acid sequences that correspond to, but are not limited to, the animal species and isotype.
  • the monoclonal antibody according to the present invention can be produced by a conventional method. For example, first, an animal is immunized with an antigen. When producing an anti-A1 beta casein antibody specific to A1 beta casein, an antigen containing histidine at position 67 of A1 beta casein is used. When producing an anti-A1/A2 beta casein antibody that binds to both A1 beta casein and A2 beta casein, an antigen that does not contain the amino acid residue at position 67 and contains an amino acid sequence common to both A1 beta casein and A2 beta casein is used. The number of amino acid residues contained in the antigen can be adjusted to, for example, 10 or more and 20 or less.
  • mice examples include mice, rats, guinea pigs, rabbits, goats, sheep, donkeys, chickens, etc.
  • the antigen can be administered, for example, by administering an injection containing the antigen to the tail or intraperitoneally.
  • hybridomas are extracted from the spleen or lymph nodes of the immunized animal and fused with myeloma cells to produce hybridomas. From the hybridomas produced, hybridomas that produce antibodies with excellent specific binding ability to the antigen are screened using a plate on which the antigen is immobilized, and then grown. The desired antibody can then be purified from the culture medium of the resulting hybridomas.
  • the binding activity of the monoclonal antibody of the present invention to A1 beta-casein, or to A1 beta-casein and A2 beta-casein, may be evaluated separately by surface plasmon resonance, biolayer interference, or the like.
  • the monoclonal antibody of the present invention can specifically bind to A1 beta-casein, or A1 beta-casein and A2 beta-casein, and can therefore be used in particular to detect A1 beta-casein.
  • a milk sample is mixed with the anti-A1 beta-casein antibody of the present invention, and A1 beta-casein contained in the milk sample is selectively bound to it for detection.
  • detection also includes “quantification.” For example, by creating a calibration curve in advance using a milk sample with a known amount and concentration of A1 beta-casein and the monoclonal antibody of the present invention, it is possible to quantify the A1 beta-casein contained in the milk sample.
  • A1 beta-casein detection method of the present invention will be explained step by step, but the present invention is not limited to the specific examples below.
  • sample pretreatment step the milk sample in which A1 beta casein is to be detected is pretreated in order to improve the detection sensitivity.
  • This step is optional, and for example, when the milk sample is directly subjected to measurement, this step does not need to be performed.
  • Pretreatment includes, for example, dilution, defatting, and sterilization of the milk sample.
  • the dilution ratio may be determined within a range in which the concentration of A1 beta casein can be determined from the ELISA measurement value using a calibration curve, and may be, for example, 1000 times or more and 100,000 times or less.
  • PBS or the like can be used for dilution.
  • Defatting may be performed according to a conventional method. Sterilization may be performed by either high-temperature sterilization or low-temperature sterilization.
  • ELISA step In this step, A1 beta casein contained in the milk sample is detected by ELISA (Enzyme-Linked Immuno Sorbent Assay).
  • ELISA Enzyme-Linked Immuno Sorbent Assay
  • direct method indirect method
  • sandwich method etc. are mainly known, but from the viewpoint of high accuracy, sandwich ELISA will be explained below.
  • Step of immobilizing capture antibody the capture antibody is immobilized on a plate or the like.
  • Plates for immobilizing antibodies are commercially available. Specifically, a capture antibody solution is added to each well of a microtiter plate made of polyvinyl chloride, polystyrene, or the like, and incubated, followed by washing.
  • the capture antibody may be an anti-A1 beta casein antibody or an anti-A1/A2 beta casein antibody. According to the inventors' experimental findings, A1 beta casein can be detected well whether the capture antibody is an anti-A1 beta casein antibody or an anti-A1/A2 beta casein antibody.
  • a detection antibody which is an anti-A1 beta casein antibody
  • the epitope of the detection antibody is different from the epitope of the capture antibody.
  • the capture antibody may be an enzyme-labeled one, or, in addition to the detection antibody, a secondary antibody that specifically binds to the detection antibody and is enzyme-labeled may be used.
  • the detection antibody can be quantified by a method that corresponds to the labeling enzyme bound to the detection antibody or secondary antibody.
  • the labeling enzyme is horseradish peroxidase (HRP)
  • HRP horseradish peroxidase
  • a coloring agent examples include 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine dihydrochloride (OPD), and 2,2'-azino-di-[3-ethyl-benzothiazoline-6 sulfonic acid] diammonium salt (ABTS), and the absorbance at a wavelength that corresponds to the coloring agent can be measured.
  • TMB 3,3',5,5'-tetramethylbenzidine
  • OPD o-phenylenediamine dihydrochloride
  • ABTS 2,2'-azino-di-[3-ethyl-benzothiazoline-6 sulfonic acid] diammonium salt
  • the labeling enzyme is alkaline phosphat
  • the proportion of A1 beta casein in the beta casein of A2 milk which is considered to have a low proportion of A1 beta casein and no harmful effects from A1 beta casein, is said to be 30% by mass or less.
  • the monoclonal antibody of the present invention can be used to detect 2% by mass or more of A1 beta casein relative to the total beta casein, making the method of detecting A1 beta casein of the present invention an excellent method of evaluating milk.
  • the proportion of A1 beta casein relative to the total beta casein the easier it is to detect A1 beta casein, and the proportion is preferably 5% by mass or more, more preferably 10% by mass or more, and even more preferably 20% by mass or more or 30% by mass or more.
  • Example 1 Production of Monoclonal Antibodies by the Lymph Node Method (1) Preparation of Antigen/Adjuvant Emulsion Two 1 mL Luer-lock glass syringes were placed on either end of a locking needle connector. The plunger was removed from the upper syringe before connection. Complete Freund's adjuvant (750 ⁇ L) was mixed well and added to the upper syringe with the plunger removed using a pipette for non-aqueous liquids. The plunger of the lower syringe was pulled back to fill the inside with adjuvant, and then the plunger was returned to its original position, and the adjuvant was returned to the upper syringe.
  • Complete Freund's adjuvant 750 ⁇ L
  • the antigen protein shown in Table 1 was dissolved in PBS to prepare a 0.5 mg/mL solution for mice and a 1 mg/mL solution for rats. Each solution (350 ⁇ L) was placed in the upper syringe, and the plunger of the lower syringe was pulled to aspirate the antigen and adjuvant into the lower syringe. The upper syringe was removed and the plunger was returned to its original position. The plunger of the lower syringe was slowly pushed up to expel air bubbles.
  • the upper syringe was connected, the syringe was held horizontally, and the plunger was moved back and forth quickly approximately 10 times to emulsify the mixture.
  • the resulting emulsion was drawn into one of the syringes, and the other syringe and needle connector were removed and a disposable needle was attached.
  • the collected lymph nodes were transferred to a steel wire mesh in a 60 mm culture dish and DMEM (10 mL) was added. The lymph nodes were crushed with a spatula through the wire mesh.
  • the cells (mainly B cells) were collected in a 50 mL tube using a pipette. The cells were centrifuged at 1500 rpm and 500 G for 5 minutes and the supernatant was removed from the cell pellet. The cells were resuspended in DMEM (10 mL). The centrifugation and resuspension procedures were repeated.
  • the number of B cells was counted using a cell counting chamber, and a 5.0 x 10 7 cell suspension was prepared in a new 50 mL tube and incubated at room temperature for 5 minutes.
  • the B cell suspension and the myeloma cell suspension were mixed and centrifuged at 1500 rpm for 5 minutes, the supernatant was removed from the cell pellet, and the cells were resuspended in ECF buffer (10 mL). The centrifugation and resuspension procedure was repeated. Both cell suspensions were centrifuged at 1500 rpm for 5 minutes, the supernatant was removed from the cell pellet, and the cells were resuspended in ECF buffer (1 mL).
  • the culture supernatant was collected from each well into a new 1.5 mL tube and stored at 4°C or -30°C.
  • Fresh medium 250 ⁇ L was added, and the cells were suspended by pipetting.
  • Storage medium 250 ⁇ L was added, mixed well, transferred to a freezing tube, and stored at ⁇ 80° C.
  • HT medium 100 ⁇ L was added, and positive wells were confirmed by ELISA 8 days later.
  • positive hybridomas were transferred to 24-well plates and subcultured for 2-3 days, then storage medium (250 ⁇ L) was added and mixed well, transferred to cryotubes, and stored at -80°C.
  • storage medium 250 ⁇ L was added and mixed well, transferred to cryotubes, and stored at -80°C.
  • hybridomas from positive clones were thawed and cultured to grow to the capacity of 3-4 75 cm2 plates. The culture supernatant containing the monoclonal antibodies was harvested when in the logarithmic growth phase.
  • the amino acid sequences of the CDRs of rat-derived anti-A1 beta casein antibody are shown in Table 2, the amino acid sequences of the CDRs of rat-derived anti-A1/A2 beta casein antibody are shown in Table 3, and the amino acid sequences of the CDRs of mouse-derived anti-A1 beta casein antibody are shown in Table 4.
  • Example 2 Quantification of A1 beta-casein by sandwich ELISA A1 milk containing only A1 beta-casein and A2 milk containing only A2 beta-casein were each diluted 100-fold and mixed to obtain the composition shown in Table 5 to prepare A1/A2 beta-casein skim milk.
  • the #27-1 anti-A1/A2 beta casein antibody was diluted with PBS as a capture antibody to prepare a 1.5 ⁇ g/100 ⁇ L solution, and added to a 96-well plate at 100 ⁇ L/well. The plate was covered and incubated overnight at 37° C. Then, the liquid was discarded, washed twice with PBS (200 ⁇ L), and 1% BSA/PBS (200 ⁇ L) was added. The plate was covered and incubated for 1 hour at 37° C. or overnight at 4° C. Then, the liquid was discarded, washed twice with PBS (200 ⁇ L), and a milk sample (100 ⁇ L) diluted 100-fold with PBS was added.
  • the plate was covered and incubated for 1.5 to 2 hours at 37° C. Then, the liquid was discarded, and washed three times with PBS (200 ⁇ L).
  • anti-A1 beta casein antibody #6-3 was diluted with 1% BSA/PBS to prepare a 1.5 ⁇ g/100 ⁇ L solution as a detection antibody, and the resulting solution (100 ⁇ L) was added.
  • the plate was covered and incubated at 37° C. for 1.5 to 2 hours. The liquid was discarded, and the plate was washed three times with PBS (200 ⁇ L).
  • the HRP-labeled secondary antibody was diluted 20,000 times with 1% BSA/PBS, and the resulting diluted solution (100 ⁇ L) was added.
  • the horizontal axis indicates the amount of A1 beta-casein per 1 ⁇ L of the milk sample before dilution.
  • an approximation equation of y -8 ⁇ 10 ⁇ 5 x 2 +0.0138x+0.1229 was obtained between the A1 beta casein content and the measured OD value, and the coefficient of determination R 2 was 0.998, which was very high.
  • A2 milk in which the influence of A1 beta casein is reduced refers to milk in which the ratio of A1 beta casein to beta casein is suppressed to 30% or less, and therefore, according to the present invention, it has been shown that the beta casein contained in milk can be sufficiently evaluated.
  • the actual measurement target milk may be defatted, diluted 10,000 times, and then subjected to ELISA measurement under the above conditions.
  • Example 3 Quantification of A1 beta-casein by sandwich ELISA ELISA measurement was performed in the same manner as in Example 2, except that #17-2 anti-A1 beta-casein antibody was used as the capture antibody instead of the anti-A1/A2 beta-casein antibody, and #6-3 anti-A1 beta-casein antibody was used as the detection antibody. The results are shown in Figure 2. As shown in Figure 2, the results obtained when anti-A1 beta casein antibody was used as the capture antibody were similar to those obtained when anti-A1/A2 beta casein antibody was used.
  • Example 4 Quantification of A1 beta casein by sandwich ELISA ELISA measurement was performed in the same manner as in Example 2, except that rHis#4-2 anti-A1 beta casein antibody was used as the capture antibody and mHis#21-8 anti-A1 beta casein antibody was used as the detection antibody.
  • the results are shown in Figure 3.
  • A1 beta casein was detectable in milk samples containing 200 ng/ ⁇ L or more of A1 beta casein before dilution and in milk samples containing 2 ⁇ 10 ⁇ 2 ng/ ⁇ L or more of A1 beta casein after 10,000-fold dilution
  • A1 beta casein was detectable in milk samples containing 1000 ng/ ⁇ L or more of A1 beta casein before dilution and in milk samples containing 1 ⁇ 10 ⁇ 1 ng/ ⁇ L or more of A1 beta casein after 10,000-fold dilution, based on the measured OD value.
  • Example 5 Quantification of A1 beta-casein by sandwich ELISA A1 milk containing only A1 beta-casein and A2 milk containing only A2 beta-casein were each diluted 100-fold and mixed to obtain the composition shown in Table 6 to prepare A1/A2 beta-casein milk. The milk samples were used as they were without being skimmed. ELISA measurements were performed in the same manner as in Example 2, except that mHis#6-3A1 beta-casein antibody was used as the detection antibody and non-skimmed milk samples were used. The results are shown in Table 6 and FIG.
  • Example 6 Quantification of A1 beta-casein by sandwich ELISA A1 milk containing only A1 beta-casein and A2 milk containing only A2 beta-casein were each diluted 100-fold and mixed to obtain the composition shown in Table 6 to prepare A1/A2 beta-casein milk. The milk samples were used as they were without being skimmed. ELISA measurements were performed in the same manner as in Example 2, except that mHis#6-3A1 beta casein antibody was used as the detection antibody and non-skimmed milk samples were used. The results are shown in Table 7 and FIG.
  • Example 7 We have prepared rat-derived anti-A1 beta casein antibody (I), rat-derived anti-A1/A2 beta casein antibody (III), and mouse-derived anti-A1 beta casein antibody (II) having the following CDR amino acid sequences. Each antibody has excellent selective binding ability to A1 beta casein or both A1 beta casein and A2 beta casein.

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