Monoclonal antibodies and methods for detecting A1 beta-casein

Abstract

The purpose of the present invention is to provide a monoclonal antibody that binds to A1 beta-casein or to both A1 beta-casein and A2 beta-casein and to provide a method for detecting A1 beta-casein using said monoclonal antibody. The monoclonal antibody according to the present invention is characterized by having a specific antigen complementarity-determining region amino acid sequence.

Description

[Technical Field]

[0001] The present invention relates to a monoclonal antibody that binds to A1 beta-casein or both A1 and A2 beta-casein, and a method for detecting A1 beta-casein using the monoclonal antibody. [Background technology]

[0002] Milk contains approximately 3.3% protein, and approximately 80% of the protein is casein, a phosphoprotein in which phosphate is attached to the side-chain hydroxyl group of serine residues. Casein is divided into alpha, beta, and kappa forms, and beta casein is further divided into A1 and A2 forms. Among beta caseins, A1 beta casein is known to cause gas, bloating, diarrhea, and stomach discomfort when consuming milk. While not yet fully recognized in Japan, the market for A2 milk, which reduces the harmful effects of A1 beta casein, has expanded in Europe and the United States in recent years. Therefore, there is a need to measure the content of A1 beta casein in milk.

[0003] The only difference between A1 and A2 beta-casein is whether the amino acid residue at position 67 is histidine or proline, and the proportion of A1 and A2 beta-casein in milk depends on the beta-casein gene of the cow. That is, 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-casein.

[0004] Therefore, it is possible to evaluate the proportion of A1 beta-casein in milk by examining the bovine beta-casein gene using PCR. However, although PCR is highly accurate, it can only be used to test a single cow and cannot test milk products produced from multiple cows. Furthermore, PCR testing is time-consuming.

[0005] Patent Document 1 describes a method for determining the amount of A1 beta-casein or A2 beta-casein in a composition using liquid chromatography-mass spectrometry (LC-MS). LC-MS allows for testing of milk products produced from multiple cows. However, LC-MS requires specialized and expensive equipment and is time-consuming and laborious due to pre-processing and other factors.

[0006] ELISA, which uses specific antibodies against a target substance in combination with an enzyme reaction, is a known method for quantifying a target substance. The ELISA method allows for the testing of milk products rather than individual bovine samples, and is simple and time-saving. ELISA kits for quantifying A1 beta-casein are commercially available (Non-Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Special Publication No. 2022-515563 [Non-patent literature]

[0008] [Non-Patent Document 1] Biosensis® Bovine A1 Beta Casein ELISA Kit, Catalog No: BEK-2364-1P [Non-patent document 2] biosensis(registered trademark) Bovine A1 β-Casein ELISA Kit, Catalog Number: BEK-2243-1P / 2P / 5P / 10P Summary of the Invention [Problem to be solved by the invention]

[0009] As mentioned above, ELISA kits for measuring the amount of A1 beta-casein in milk are commercially available. However, these kits cannot accurately quantify A1 beta-casein in milk samples containing coexisting A2 beta-casein, for example, in milk samples containing 1 part by mass or more of A2 beta-casein relative to A1 beta-casein, because the A2 beta-casein inhibits accurate measurement. Therefore, 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. [Means for solving the problem]

[0010] 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 and A2 beta-casein, thereby completing the present invention. The present invention will now be described.

[0011] [1] 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. [2] 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. [3] 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. [4] The monoclonal antibody according to any one of [1] to [3] above, further comprising an Fc region.

[0012] [5] A hybridoma comprising nucleic acids encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody according to any one of [1] to [3] above.

[0013] [6] A method for detecting A1 beta-casein in a milk sample, comprising: 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 described in [1] or [2] above, and selecting a capture antibody having an epitope different from that of the detection antibody from the monoclonal antibodies described in any one of [1] to [3] above; Immobilizing the capture antibody on a plate; and The method according to [6] above, which comprises a step of mixing the detection antibody with the mixture of the capture antibody and the milk sample. [Effects of the Invention]

[0014] The monoclonal antibody of the present invention can specifically bind to A1 beta-casein or both A1 beta-casein and A2 beta-casein. Therefore, 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 industrially very advantageous as a technology that can easily evaluate milk, focusing on A1 beta-casein, which has been attracting attention in recent years. [Brief explanation of the drawings]

[0015] [Figure 1] 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 according to the present invention. [Figure 2] FIG. 2 is a graph showing the results of quantifying A1 beta-casein in milk samples by sandwich ELISA using the anti-A1 beta-casein antibody according to the present invention. [Figure 3] FIG. 3 is a graph showing the results of quantifying A1 beta-casein in milk samples by sandwich ELISA using the anti-A1 beta-casein antibody according to the present invention. [Figure 4] 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 according to the present invention. [Figure 5] 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 according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0016] Hereinafter, the anti-A1 beta-casein monoclonal antibody obtained by the inventors from rat cells will 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.

[0017] (1) Monoclonal antibody (I) Monoclonal antibody (I) comprises a heavy chain variable region comprising a heavy chain complementarity determining region 1 (VH CDR1) comprising the amino acid sequence of SEQ ID NO: 301, a heavy chain complementarity determining region 2 (VH CDR2) comprising the amino acid sequence of SEQ ID NO: 302, and a 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 a light chain complementarity determining region 1 (VL CDR1) comprising the amino acid sequence of SEQ ID NO: 304, a light chain complementarity determining region 2 (VL CDR2) comprising the amino acid sequence of SEQ ID NO: 305, and a light chain complementarity determining region 3 (VL CDR3) comprising the amino acid sequence of SEQ ID NO: 306; and binds to A1 beta-casein.

[0018] SEQ ID NO: 301: Xaa 1 -Xaa 2 -Xaa 3 -Xaa 4 -Xaa 5 -Xaa 6 -Xaa 7 -Xaa 8 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 / Gln,Xaa 8 =Asp / Glu / Gly / Ala / Thr / Ser / Phe / Tyr / Trp

[0019] SEQ ID NO: 301 includes SEQ ID NO: 501 below. SEQ ID NO: 501: Xaa 701 -Xaa 702 -Xaa 703 -Xaa 704 -Xaa 705 -Xaa 706 -Xaa 707 -Xaa 708 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

[0020] As SEQ ID NO: 301, the following SEQ ID NOs: 336 to 344 are preferred. SEQ ID NO: 336: Xaa 299 -Xaa 300 -Xaa 301 -Xaa 302 -Xaa 303 -Xaa 304 -Xaa 305 -Xaa 306 Xaa 299 = Gly / Ala, Xaa 300 = Tyr / Phe / Trp,Xaa 301=Thr / Ser,Knock 302 =Tyr / Phe / Trp,Xaa 303 =Thr / Ser,Knock 304 =Asn / Gln,Xa 305 =Tyr / Phe / Trp,Xaa 306 =Asp / Glu

[0021] 337: Yes 307 -No 308 -No 309 -No 310 -No 311 -No 312 -No 313 -No 314 Yeah 307 =Gly / Ala,No 308 =Tyr / Phe / Trp,Xaa 309 =Thr / Ser,Knock 310 =Tyr / Phe / Trp,Xaa 311 =Thr / Ser,Knock 312 =Asn / Gln,Xa 313 =Tyr / Phe / Trp,Xaa 314 =Gly / Ala

[0022] 338: Yes 315 -No 316 -No 317 -No 318 -No 319 -No 320 -No 321 -No 322 Yeah 315 =Gly / Ala,No 316 =Tyr / Phe / Trp,Xaa 317 =Thr / Ser,Knock 318 =Tyr / Phe / Trp,Xaa 319 =Thr / Ser,Knock 320 =Thr / Ser,Knock 321 =Tyr / Phe / Trp,Xaa 322 =Gly / Ala

[0023] 339: Yes 323 -No 324 -No 325 -No 326 -No 327 -No 328 -No 329 -No 330 Yeah 323 =Gly / Ala,No 324 =Tyr / Phe / Trp,Xaa 325 =Thr / Ser,Knock 326 =Tyr / Phe / Trp,Xaa 327 =Lys / Arg / His, Rock 328 =Asn / Gln,Xa 329 =Tyr / Phe / Trp,Xaa 330 =Asp / Glu

[0024] 340: Yes 331 -No 332 -No 333 -No 334 -No 335 -No 336 -No 337 -No 338 Yeah 331 =Gly / Ala,No 332 =Tyr / Phe / Trp,Xaa 333 =Thr / Ser,Knock 334 =Tyr / Phe / Trp,Xaa 335 =Thr / Ser,Knock 336 =Thr / Ser,Knock 337 =Tyr / Phe / Trp,Xaa 338 =Thr / Ser

[0025] 341: Yes 339 -No 340 -No 341 -No 342 -No 343 -No 344 -No 345 -No 346 Yeah 339=Gly / Ala,No 340 =Tyr / Phe / Trp,Xaa 341 =Val / Lion / Dog,Dog 342 =Tyr / Phe / Trp,Xaa 343 =Arg / Lys / His,stone 344 =Asn / Gln,Xa 345 =Tyr / Phe / Trp,Xaa 346 =Gly / Ala

[0026] 342: Yes 347 -No 348 -No 349 -No 350 -No 351 -No 352 -No 353 -No 354 Yeah 347 =Gly / Ala,No 348 =Tyr / Phe / Trp,Xaa 349 =Thr / Ser,Knock 350 =Tyr / Phe / Trp,Xaa 351 =Thr / Ser,Knock 352 =Asn / Gln,Xa 353 =Tyr / Phe / Trp,Xaa 354 =Tyr / Phe / Trp

[0027] 343: Yes 355 -No 356 -No 357 -No 358 -No 359 -No 360 -No 361 -No 362 Yeah 355 =Gly / Ala,No 356 =Tyr / Phe / Trp,Xaa 357 =Thr / Ser,Knock 358 =Tyr / Phe / Trp,Xaa 359 =Thr / Ser,Knock 360 =Asn / Gln,Xa 361 =Tyr / Phe / Trp,Xaa362 =Gly / Ala

[0028] 344: Yes 363 -No 364 -No 365 -No 366 -No 367 -No 368 -No 369 -No 370 Yeah 363 =Gly / Ala,No 364 =Tyr / Phe / Trp,Xaa 365 =Thr / Ser,Knock 366 =Tyr / Phe / Trp,Xaa 367 =Asp / Glu,Xa 368 =Asp / Glu,Xa 369 =Tyr / Phe / Trp,Xaa 370 =Gly / Ala

[0029] 302: Yes 9 -No 10 -No 11 -No 12 -No 13 -No 14 -No 15 -No 16 Yeah 9 =Ile / Val / Leu / Phe / Tyr / Trp,Xaa 10 =Thr / Ser / Asn / Gln / Lys / Arg / His / Gly / Ala / Tyr / Phe / Trp,Xaa 11 =Thr / Ser / Pro / Tyr / Phe / Trp / Gly / Ala / Asn / Gln / Val / Leu / Ile / Asp / Glu,Xaa 12 =Ser / Thr / Asp / Glu / Lys / Arg / His / Phe / Tyr / Trp / Gly / Ala / Asn / Gln,Xaa 13 =Gly / Ala / Ser / Thr / Asn / Gln / Lys / Arg / His / Asp / Glu,Xaa 14 =Gly / Ala / Ser / Thr / Val / Leu / Ile / Lys / Arg / His,Xaa15 =Ser / Thr / Tyr / Phe / Trp / Lys / Arg / His / Gly / Ala / Asn / Gln / Val / Leu / Ile / Asp / Glu / Cys / Met,Xaa 16 =Asp / Glu / Gly / Ala / Thr / Ser / Pro / Val / Leu / Ile

[0030] Examples of SEQ ID NO: 302 include SEQ ID NO: 502 below. SEQ ID NO:502: Xaa 709 -Xaa 710 -Xaa 711 -Xaa 712 -Xaa 713 -Xaa 714 -Xaa 715 -Xaa 716 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

[0031] As SEQ ID NO: 302, the following SEQ ID NOs: 345 to 353 are preferred. SEQ ID NO: 345: Xaa 371 -Xaa 372 -Xaa 373 -Xaa 374 -Xaa 375 -Xaa 376 -Xaa 377 -Xaa 378 Xaa 371 =Val / Leu / Ile,Xaa 372 =Thr / Ser,Xaa 373 =Thr / Ser,Xaa374 =Thr / Ser,Knock 375 =Gly / Ala,No 376 =Gly / Ala,No 377 =Thr / Ser,Knock 378 =Thr / Ser

[0032] 346: Yes 379 -No 380 -No 381 -No 382 -No 383 -No 384 -No 385 -No 386 Yeah 379 =Val / Lion / Dog,Dog 380 =Asn / Gln,Xa 381 =Tyr / Phe / Trp,Xaa 382 =Asp / Glu,Xa 383 =Gly / Ala,No 384 =Thr / Ser,Knock 385 =Thr / Ser,Knock 386 =Thr / Ser

[0033] 347: Yes 387 -No 388 -No 389 -No 390 -No 391 -No 392 -No 393 -No 394 Yeah 387 =Val / Lion / Dog,Dog 388 =Thr / Ser,Knock 389 =Pro,Yes 390 =Thr / Ser,Knock 391 =Gly / Ala,No 392 =Gly / Ala,No 393 =Thr / Ser,Knock 394 =Val / Lion / Home

[0034] 348: Yes 395 -No 396-No 397 -No 398 -No 399 -No 400 -No 401 -No 402 Yeah 395 =Val / Lion / Dog,Dog 396 =Asn / Gln,Xa 397 =Pro,Yes 398 =Thr / Ser,Knock 399 =Thr / Ser,Knock 400 =Gly / Ala,No 401 =Tyr / Phe / Trp,Xaa 402 =Val / Lion / Home

[0035] 349: Yes 403 -No 404 -No 405 -No 406 -No 407 -No 408 -No 409 -No 410 Yeah 403 =Val / Lion / Dog,Dog 404 =Asn / Gln,Xa 405 =Pro,Yes 406 =Thr / Ser,Knock 407 =Thr / Ser,Knock 408 =Gly / Ala,No 409 =Tyr / Phe / Trp,Xaa 410 =Thr / Ser

[0036] 350: Yes 411 -No 412 -No 413 -No 414 -No 415 -No 416 -No 417 -No 418 Yeah 411 =Tyr / Phe / Trp,Xaa 412 =Asn / Gln,Xa 413 =Pro,Yes414 =Thr / Ser,Knock 415 =Thr / Ser,Knock 416 =Gly / Ala,No 417 =Tyr / Phe / Trp,Xaa 418 =Thr / Ser

[0037] 351: Yes 419 -No 420 -No 421 -No 422 -No 423 -No 424 -No 425 -No 426 Yeah 419 =Val / Lion / Dog,Dog 420 =Asn / Gln,Xa 421 =Thr / Ser,Knock 422 =Tyr / Phe / Trp,Xaa 423 =Thr / Ser,Knock 424 =Gly / Ala,No 425 =Asp / Glu,Xa 426 =Thr / Ser

[0038] 352: Yes 427 -No 428 -No 429 -No 430 -No 431 -No 432 -No 433 -No 434 Yeah 427 =Val / Lion / Dog,Dog 428 =Thr / Ser,Knock 429 =Asn / Gln,Xa 430 =Gly / Ala,No 431 =Gly / Ala,No 432 =Gly / Ala,No 433 =Thr / Ser,Knock 434 =Thr / Ser

[0039] 353: Yes 435 -No436 -Xaa 437 -Xaa 438 -Xaa 439 -Xaa 440 -Xaa 441 -Xaa 442 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

[0040] SEQ ID NO: 303: Xaa 17 -Xaa 18 -Xaa 19 -Xaa 20 -Xaa 21 -Xaa 22 -Xaa 23 -Xaa 24 -Xaa 25 -Xaa 26 -Xaa 27 -Xaa 28 -Xaa 29 -Xaa 30 -Xaa 31 -Xaa 32 -Xaa 33 -Xaa 34 -Xaa 35 -Xaa 36 -Xaa 37 Xaa 17 =Ala / Gly / Deletion, Xaa 18 =Arg / Lys / His / Ser / Thr, Xaa 19 =His / Arg / Lys / Deletion, Xaa 20 =Pro / Gly / Ala / Ser / Thr / Asp / Glu, Xaa 21 =Tyr / Phe / Trp / Arg / Lys / His / Gly / Ala / Deletion, Xaa 22=Asn / Gln / Tyr / Phe / Trp / Asp / Glu / Arg / Lys / His / deletion,Xaa 23 =Asn / Gln / Val / Leu / Ile / deletion, Xaa 24 =Tyr / Phe / Trp / Val / Leu / Ile / deletion,Xaa 25 =Asp / Glu / Gly / Ala / Deletion, Xaa 26 = Gly / Ala / deletion, Xaa 27 = Ser / Thr / Asn / Gln / deletion, Xaa 28 = Tyr / Phe / Trp / deletion, Xaa 29 =Tyr / Phe / Trp / Asn / Gln / Ser / Thr / deletion, Xaa 30 =Arg / Lys / His / Asp / Glu / deletion,Xaa 31 =Tyr / Phe / Trp / Gly / Ala / deletion,Xaa 32 =Gly / Ala / Tyr / Phe / Trp / Pro / Ser / Thr / deletion,Xaa 33 = Gly / Ala / Tyr / Phe / Trp / deletion, Xaa 34 =Asp / Glu / Ser / Thr / Tyr / Phe / Trp / Arg / Lys / His / Val / Leu / Ile / deletion,Xaa 35 = Phe / Tyr / Trp / deletion, Xaa 36 = Asp / Glu / Gly / Ala, Xaa 37 =Phe / Tyr / Trp

[0041] Examples of SEQ ID NO: 303 include SEQ ID NO: 503 below. SEQ ID NO:503: Xaa 717 -Xaa 718 -Xaa 719 -Xaa 720 -Xaa 721 -Xaa 722 -Xaa 723 -Xaa 724 -Xaa 725 -Xaa 726 -Xaa 727 -Xaa 728 -Xaa 729 -Xaa 730 -Xaa 731 -Xaa732 -Xaa 733 -Xaa 734 -Xaa 735 -Xaa 736 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,Xaa 722 =Asn / Gln / Deletion,Xaa 723 =Asn / Gln / Deletion,Xaa 724 =Tyr / Phe / Trp,Xaa 725 =Asp / Glu / Deletion,Xaa 726 =Gly / Ala,Xaa 727 =Ser / Thr / Asn / Gln / Deletion,Xaa 728 =Tyr / Phe / Trp,Xaa 729 =Tyr / Phe / Trp,Xaa 730 =Tyr / Phe / Trp,Xaa 731 =Gly / Ala / Deletion,Xaa 732 =Gly / Ala / Deletion,Xaa 733 =Asp / Glu / Ser / Thr / Deletion,Xaa 734 =Phe / Tyr / Trp,Xaa 735 =Asp / Glu,Xaa 736 =Phe / Tyr / Trp

[0042] SEQ ID NO: 303 preferably includes the following SEQ ID NOs: 315, 316, 354 to 356. SEQ ID NO: 315: Xaa 126 -Xaa 127 -Xaa 128 -Xaa 129 -Xaa 130 -Xaa 131 -Xaa 132 -Xaa 133 -Xaa 134 -Xaa 135 -Xaa 136 -Xaa 137 -Xaa 138-No 139 Yeah 126 =Ala / Gly,Rock 127 =Arg / Lys / His,stone 128 =Pro / Gly / Wing,Dog 129 =Tyr / Phe / Trp,Xaa 130 =Asn / Gln,Xa 131 =Asn / Gln,Xa 132 =Tyr / Phe / Trp,Xaa 133 =Gly / Ala,No 134 =Tyr / Phe / Trp,Xaa 135 =Tyr / Phe / Trp,Xaa 136 =Tyr / Phe / Trp,Xaa 137 =Phe / Tyr / Trp,Xaa 138 =Asp / Glu,Xa 139 =Phe / Tyr / Trp

[0043] 316: Yes 140 -No 141 -No 142 -No 143 -No 144 -No 145 -No 146 -No 147 -No 148 -No 149 -No 150 -No 151 -No 152 -No 153 -No 154 -No 155 -No 156 -No 157 Yeah 140 =Ala / Gly,Rock 141 =Arg / Lys / His,stone 142 =His / Arg / Lys, Rock 143 =Pro / Gly / Ala / Cer / Thr,Dog 144 =Tyr / Phe / Trp,Xaa 145 =Tyr / Phe / Trp,Xaa 146 =Asp / Glu,Xa 147=Gly / Ala,No 148 =Ser / Thr / Asn / Gln,Xa 149 =Tyr / Phe / Trp,Xaa 150 =Tyr / Phe / Trp,Xaa 151 =Tyr / Phe / Trp,Xaa 152 =Gly / Ala,No 153 =Gly / Ala,No 154 =Asp / Glu / Ser / Thr,Da 155 =Phe / Tyr / Trp,Xaa 156 =Asp / Glu,Xa 157 =Phe / Tyr / Trp

[0044] 354: Yes 443 -No 444 -No 445 -No 446 -No 447 -No 448 -No 449 -No 450 -No 451 -No 452 -No 453 -No 454 -No 455 -No 456 -No 457 -No 458 -No 459 -No 460 Yeah 443 =Gly / Ala,No 444 =His / Arg / Lys, Rock 445 =His / Arg / Lys, Rock 446 =Thr / Ser,Knock 447 =Tyr / Phe / Trp,Xaa 448 =Tyr / Phe / Trp,Xaa 449 =Asp / Glu,Xa 450 =Gly / Ala,No 451 =Asn / Gln,Xa 452 =Tyr / Phe / Trp,Xaa 453 =Tyr / Phe / Trp,Xaa 454 =Tyr / Phe / Trp,Xaa455 =Gly / Ala,No 456 =Gly / Ala,No 457 =Cer / Thr,No 458 =Tyr / Phe / Trp,Xaa 459 =Asp / Glu,Xa 460 =Tyr / Phe / Trp

[0045] 355: Yes 461 -No 462 -No 463 -No 464 -No 465 -No 466 -No 467 -No 468 -No 469 -No 470 -No 471 -No 472 -No 473 -No 474 -No 475 -No 476 -No 477 -No 478 Yeah 461 =Gly / Ala,No 462 =His / Arg / Lys, Rock 463 =His / Arg / Lys, Rock 464 =Gly / Ala,No 465 =Tyr / Phe / Trp,Xaa 466 =Tyr / Phe / Trp,Xaa 467 =Asp / Glu,Xa 468 =Gly / Ala,No 469 =Cer / Thr,No 470 =Tyr / Phe / Trp,Xaa 471 =Tyr / Phe / Trp,Xaa 472 =Tyr / Phe / Trp,Xaa 473 =Gly / Ala,No 474 =Gly / Ala,No 475 =Asp / Glu,Xa 476 =Tyr / Phe / Trp,Xaa 477 =Asp / Glu,Xa 478=Tyr / Phe / Trp

[0046] SEQ ID NO: 356: Xaa 479 -Xaa 480 -Xaa 481 -Xaa 482 -Xaa 483 -Xaa 484 -Xaa 485 -Xaa 486 -Xaa 487 -Xaa 488 -Xaa 489 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

[0047] 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 [[ID=八十二]]=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, Xaa 45 = Gly / Ala / deletion, Xaa 46 =Tyr / Phe / Trp / Asn / Gln / Lys / Arg / His / deletion,Xaa 47 =Lys / Arg / His / Ser / Thr / deletion,Xaa 48 =Asn / Gln / Tyr / Phe / Trp / deletion

[0048] Examples of SEQ ID NO: 304 include SEQ ID NO: 504 below. SEQ ID NO:504: Xaa 737 -Xaa 738 -Xaa 739 -Xaa 740 -Xaa 741 -Xaa 742 -Xaa 743 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

[0049] As SEQ ID NO: 304, the following SEQ ID NOs: 317, 318, and 357 to 359 are preferred. SEQ ID NO: 317: Xaa 158 -Xaa 159 -Xaa 160 -Xaa 161 -Xaa 162 -Xaa 163 Xaa 158 =Gln / Asn,Xaa 159 =Asn / Gln,Xaa 160=Home / Val / Lion,Dog 161 =Tyr / Phe / Trp,Xaa 162 =Lys / Arg / His, Rock 163 =Asn / Gln

[0050] 318: Yes 164 -No 165 -No 166 -No 167 -No 168 Yeah 164 =Cer / Thr,No 165 =Ser / Thr / Arg / Lys / His,Xa 166 =Home / Val / Lion,Dog 167 =Cer / Thr,No 168 =Tyr / Phe / Trp

[0051] 357: Yes 490 -No 491 -No 492 -No 493 -No 494 Yeah 490 =Cer / Thr,No 491 =His / Arg / Lys, Rock 492 =Home / Val / Lion,Dog 493 =Cer / Thr,No 494 =Tyr / Phe / Trp

[0052] 358: Yes 495 -No 496 -No 497 -No 498 -No 499 Yeah 495 =Cer / Thr,No 496 =Cer / Thr,No 497 =Home / Val / Lion,Dog 498 =Cer / Thr,No 499 =Tyr / Phe / Trp

[0053] 359: Yes500 -Xaa 501 -Xaa 502 -Xaa 503 -Xaa 504 -Xaa 505 Xaa 500 =Asn / Gln,Xaa 501 =Asn / Gln,Xaa 502 =Ile / Val / Leu,Xaa 503 =Asn / Gln,Xaa 504 =Asn / Gln,Xaa 505 =Tyr / Phe / Trp

[0054] SEQ ID NO: 305: Xaa 49 -Xaa 50 -Xaa 51 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

[0055] Examples of SEQ ID NO: 305 include SEQ ID NO: 505 below. 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

[0056] As SEQ ID NO: 305, the following SEQ ID NOs: 319, 320 and 360 are preferred. SEQ ID NO: 319: Xaa 169 -Xaa 170 -Xaa 171 Xaa 169 =Gln / Asn,Xaa 170 = Ala / Gly,Xaa171 = Gln / Asn

[0057] SEQ ID NO: 320: Xaa 172 - Xaa 173 - Xaa 174 Xaa 172 = Asp / Glu, Xaa 173 = Thr / Ser, Xaa 174 = Ser / Thr

[0058] SEQ ID NO: 360: Xaa 506 - Xaa 507 - Xaa 508 Xaa 506 = Tyr / Phe / Trp, Xaa 507 = Aly / Gly, Xaa 508 = Ser / Thr

[0059] SEQ ID NO: 306: Xaa 52 - Xaa 53 - Xaa 54 - Xaa 55 - Xaa 56 - Xaa 57 - Xaa 58 - Xaa 59 - Xaa 60 - Xaa 61 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, Xaa 57 = Gln / Asn / Ser / Thr / Gly / Ala / Tyr / Phe / Trp / Ile / Val / Leu / Deletion, Xaa 58 = Thr / Ser / Pro / Deletion, Xaa59 = Gly / Ala / Pro / Gln / Asn / deletion, Xaa 60 =Leu / Val / Ile / Tyr / Phe / Trp / deletion,Xaa 61 =Thr / Ser

[0060] Examples of SEQ ID NO: 306 include SEQ ID NO: 506 below. SEQ ID NO:506: Xaa 747 -Xaa 748 -Xaa 749 -Xaa 750 -Xaa 751 -Xaa 752 -Xaa 753 -Xaa 754 -Xaa 755 -Xaa 756 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,Xaa 756 =Thr / Ser

[0061] As SEQ ID NO: 306, the following SEQ ID NOs: 321, 322, and 361 to 363 are preferred. SEQ ID NO: 321: Xaa 175 -Xaa 176 -Xaa 177 -Xaa 178 -Xaa 179 -Xaa 180 -Xaa 181 -Xaa 182 Xaa 175 =Gln / Asn,Xaa 176 =Gln / Asn,Xaa 177 = Tyr / Phe / Trp,Xaa178 =Tyr / Phe / Trp,Xaa 179 =Cer / Thr,No 180 =Gly / Ala / Pro,Dog 181 =Lion / Val / Dog,Dog 182 =Thr / Ser

[0062] 322: Yes 183 -No 184 -No 185 -No 186 -No 187 -No 188 -No 189 -No 190 -No 191 Yeah 183 =Gln / Asn,Xa 184 =Gln / Asn,Xa 185 =Tyr / Phe / Trp,Xaa 186 =Cer / Thr,No 187 =Gln / Asn / Cer / Thr,Xa 188 =Thr / Ser,Knock 189 =Gly / Ala / Pro,Dog 190 =Tyr / Phe / Trp,Xaa 191 =Thr / Ser

[0063] 361: Yes 509 -No 510 -No 511 -No 512 -No 513 -No 514 -No 515 -No 516 -No 517 Yeah 509 =Gln / Asn,Xa 510 =Gln / Asn,Xa 511 =Tyr / Phe / Trp,Xaa 512 =Cer / Thr,No 513 =Cer / Thr,No 514 =Cer / Thr,No 515 =Pro,Yes 516=Tyr / Phe / Trp,Xaa 517 =Ser / Thr

[0064] 362: Yes 518 -No 519 -No 520 -No 521 -No 522 -No 523 -No 524 -No 525 -No 526 Yeah 518 =Gln / Asn,Xa 519 =Gln / Asn,Xa 520 =Tyr / Phe / Trp,Xaa 521 =Cer / Thr,No 522 =Gln / Asn,Xa 523 =Cer / Thr,No 524 =Pro,Yes 525 =Tyr / Phe / Trp,Xaa 526 =Ser / Thr

[0065] 363: Yes 527 -No 528 -No 529 -No 530 -No 531 -No 532 -No 533 -No 534 Yeah 527 =Gln / Asn,Xa 528 =Gln / Asn,Xa 529 =Tyr / Phe / Trp,Xaa 530 =Gln / Asn,Xa 531 =Cer / Thr,No 532 =Tyr / Phe / Trp,Xaa 533 =Lion / Val / Dog,Dog 534 =Ser / Thr

[0066] (2) Click here (II) 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.

[0067] 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,Xaa 66 =Gly / Ala / Lys / Arg / His / Asp / Glu / Pro / Val / Leu / Ile / Tyr / Phe / Trp / deletion,Xaa 67 =Tyr / Phe / Trp / Lys / Arg / His / Ser / Thr / deletion,Xaa 68 =Tyr / Phe / Trp / Asn / Gln / Pro / Gly / Ala / Lys / Arg / His / deletion,Xaa 69=Gly / Ala / Tyr / Phe / Trp / Asp / Glu / Ser / Thr / Deletion, Xaa 70 =Asn / Gln / Ala / Gly / Ser / Thr / Phe / Tyr / Trp / Asp / Glu / Lys / Arg / His / Deletion, Xaa 71 =Phe / Tyr / Trp / Ser / Thr / Val / Leu / Ile / Asp / Glu / Ala / Gly / Deletion, Xaa 72 =Tyr / Phe / Trp / Asp / Glu / Lys / Arg / His / Ser / Thr / Gly / Ala / Pro, Xaa 73 =Val / Leu / Ile / Asp / Glu / Ser / Thr / Cys / Met / Lys / Arg / His / Gly / Ala / Deletion, Xaa 74 =Tyr / Phe / Trp / Gly / Ala / Ser / Thr / Deletion, Xaa 75 =Pro / Val / Leu / Ile / Gly / Ala / Phe / Tyr / Trp / Deletion, Xaa 76 =Phe / Tyr / Trp / Met / Cys / Val / Leu / Ile / Lys / Arg / His / Deletion, Xaa 77 =Asp / Glu / Gly / Ala / Pro / Val / Leu / Ile, Xaa 78 =Tyr / Phe / Trp

[0068] 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<00,00879>-Xaa<00008,80>-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,Dog 761 =Gly / Ala / Lys / Arg / His,Xa 762 =Tyr / Phe / Trp / Asp / Glu,Xaa 763 =Tyr / Phe / Trp,Xaa 764 =Pro / Val / Lion / Home,Dog 765 =Phe / Tyr / Trp / Met / Cys,Xaa 766 =Asp / Glu,Xa 767 =Tyr / Phe / Trp,Xaa 768 =Phe / Tyr / Trp

[0069] The 307's 323 and 324's 364~366's 364~3 323: Yes 192 -No 193 -No 194 -No 195 -No 196 -No 197 -No 198 -No 199 -No 200 -No 201 -No 202 Yeah 192 =Ala / Gly,Rock 193 =Val / Lion / Dog,Dog 194 =Tyr / Phe / Trp,Xaa 195 =Asp / Glu,Xa 196 =Gly / Ala,No 197 =Tyr / Phe / Trp,Xaa 198 =Tyr / Phe / Trp,Xaa 199 =Pro / Val / Lion / Home,Dog 200 =Phe / Tyr / Trp,Xaa 201 =Asp / Glu,Xa 202 =Tyr / Phe / Trp

[0070] 324: Yes 203 -No 204 -No 205 -No 206 -No207 -No 208 -No 209 -No 210 -No 211 -No 212 -No 213 Yeah 203 =Ala / Gly,Rock 204 =Gly / Ala,No 205 =Tyr / Phe / Trp,Xaa 206 =Ala / Gly,Rock 207 =Lys / Arg / His, Rock 208 =Asp / Glu,Xa 209 =Tyr / Phe / Trp,Xaa 210 =Pro / Val / Lion / Home,Dog 211 =Met / Cys, Rock 212 =Asp / Glu,Xa 213 =Tyr / Phe / Trp

[0071] 364: Yes 535 -No 536 -No 537 -No 538 -No 539 -No 540 -No 541 -No 542 -No 543 -No 544 -No 545 Yeah 535 =Gly / Ala,No 536 =Lys / Arg / His, Rock 537 =Gln / Asn,Xa 538 =Tyr / Phe / Trp,Xaa 539 =Tyr / Phe / Trp,Xaa 540 =Gly / Ala,No 541 =Val / Lion / Dog,Dog 542 =Cer / Thr,No 543 =Gly / Ala,No 544 =Asp / Glu,Xa 545 =Tyr / Phe / Trp

[0072] 365: Yes 546 -No 547 -No 548 -No 549 -No 550 -No 551 -No 552 -No 553 -No 554 -No 555 -No 556 -No 557 -No 558 -No 559 -No 560 -No 561 Yeah 546 =Gly / Ala,No 547 =Lys / Arg / His, Rock 548 =Lys / Arg / His, Rock 549 =Gly / Ala,No 550 =Val / Lion / Dog,Dog 551 =Tyr / Phe / Trp,Xaa 552 =Gln / Asn,Xa 553 =Tyr / Phe / Trp,Xaa 554 =Asp / Glu,Xa 555 =Val / Lion / Dog,Dog 556 =Gly / Ala,No 557 =Cer / Thr,No 558 =Tyr / Phe / Trp,Xaa 559 =Tyr / Phe / Trp,Xaa 560 =Gly / Ala,No 561 =Tyr / Phe / Trp

[0073] 366: Yes 562 -No 563 -No 564 -No 565 -No 566 -No 567 -No 568 -No 569 -No 570 -No 571 -No 572 -No 573 -No574 -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 571 =Tyr / Phe / Trp,Xaa 572 =Gly / Ala,Xaa<� 573 =Cys / Met,Xaa 574 =Gly / Ala,Xaa 575 =Tyr / Phe / Trp,Xaa 576 =Tyr / Phe / Trp,Xaa 577 =Gly / Ala,Xaa 578 =Tyr / Phe / Trp

[0074] SEQ ID NO: 308: Xaa 79 -Xaa 80 -Xaa 81 -Xaa 82 -Xaa 83 -Xaa 84 -Xaa 85 -Xaa 86 -Xaa 87 -Xaa 88 -Xaa 89 -Xaa 90 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 It should be noted that there is a "�" in the original text which seems to be an incorrect character. I have translated it as it is but it might need to be corrected in the original source.=Asn / Gln / Arg / Lys / His / Ser / Thr / Asp / Glu / deletion, Xaa 84 =Ser / Thr / Leu / Val / Ile / deletion,Xaa 85 = Arg / Lys / His / Gly / Ala / Gln / Asn / Asp / Glu / deletion, Xaa 86 = Thr / Ser / Gly / Ala / deletion, Xaa 87 = Arg / Lys / His / Gln / Asn / deletion, Xaa 88 =Lys / Arg / His / Tyr / Phe / Trp / deletion,Xaa 89 =Asn / Gln / Ser / Thr / deletion, Xaa 90 =Tyr / Phe / Trp

[0075] Examples of SEQ ID NO: 308 include SEQ ID NO: 508 below. SEQ ID NO:508: Xaa 768 -Xaa 769 -Xaa 770 -Xaa 771 -Xaa 772 -Xaa 773 -Xaa 774 -Xaa 775 -Xaa 776 -Xaa 777 -Xaa 778 -Xaa 779 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,Xaa 778 =Asn / Gln,Xaa 779 =Tyr / Phe / Trp

[0076] The shape of the 308 should be the same as the shape of the 367~371. 367: Yes 579 -No 580 -No 581 -No 582 -No 583 -No 584 -No 585 -No 586 -No 587 -No 588 -No 589 -No 590 Yeah 579 =Gln / Asn,Xa 580 =Cer / Thr,No 581 =Val / Lion / Dog,Dog 582 =Val / Lion / Dog,Dog 583 =Gln / Asn,Xa 584 =Cer / Thr,No 585 =Arg / Lys / His,stone 586 =Cer / Thr,No 587 =Arg / Lys / His,stone 588 =Arg / Lys / His,stone 589 =Gln / Asn,Xa 590 =Tyr / Phe / Trp

[0077] 368: Yes 591 -No 592 -No 593 -No 594 -No 595 -No 596 -No 597 -No 598 -No 599 -No 600 -No 601 Yeah 591 =Gln / Asn,Xa 592 =Gly / Ala,No 593 =Val / Lion / Dog,Dog 594 =Val / Lion / Dog,Dog 595 =Arg / Lys / His,stone 596=Cer / Thr,No 597 =Gln / Asn,Xa 598 =Gly / Ala,No 599 =Gln / Asn,Xa 600 =Cer / Thr,No 601 =Tyr / Phe / Trp

[0078] 369: Yes 602 -No 603 -No 604 -No 605 -No 606 -No 607 -No 608 -No 609 -No 610 -No 611 Yeah 602 =Arg / Lys / His,stone 603 =Cer / Thr,No 604 =Val / Lion / Dog,Dog 605 =Cer / Thr,No 606 =Cer / Thr,No 607 =Cer / Thr,No 608 =Gly / Ala,No 609 =Tyr / Phe / Trp,Xaa 610 =Cer / Thr,No 611 =Tyr / Phe / Trp

[0079] 370: Yes 612 -No 613 -No 614 -No 615 -No 616 -No 617 -No 618 -No 619 -No 620 -No 621 -No 622 Yeah 612 =Asn / Gln,Xa 613 =Cer / Thr,No 614 =Val / Lion / Dog,Dog 615 =Val / Lion / Dog,Dog 616=Arg / Lys / His,stone 617 =Cer / Thr,No 618 =Asn / Gln,Xa 619 =Gly / Ala,No 620 =Asn / Gln,Xa 621 =Cer / Thr,No 622 =Tyr / Phe / Trp

[0080] 371: Yes 623 -No 624 -No 625 -No 626 -No 627 -No 628 -No 629 -No 630 -No 631 -No 632 -No 633 Yeah 623 =Asn / Gln,Xa 624 =Gly / Ala,No 625 =Val / Lion / Dog,Dog 626 =Val / Lion / Dog,Dog 627 =Arg / Lys / His,stone 628 =Cer / Thr,No 629 =Asn / Gln,Xa 630 =Gly / Ala,No 631 =Asn / Gln,Xa 632 =Cer / Thr,No 633 =Tyr / Phe / Trp

[0081] 309: Yes 91 -No 92 -No 93 Yeah 91 =Trp / Phe / Tyr / Lys / Arg / His / Gly / Ala / Leu / Val / Ile,Xaa 92 =Ala / Gly / Leu / Val / Ile / Ser / Thr,Zaa 93 =Ser / Thr

[0082] Examples of SEQ ID NO: 309 include SEQ ID NO: 509 below. SEQ ID NO: 509: Xaa 780 -Xaa 781 -Xaa 782 Xaa 780 =Trp / Phe / Tyr,Xaa 781 = Ala / Gly,Xaa 782 =Ser / Thr

[0083] As SEQ ID NO: 309, the following SEQ ID NOs: 372 to 374 are preferred. SEQ ID NO: 372: Xaa 634 -Xaa 635 -Xaa 636 Xaa 634 = Tyr / Phe / Trp,Xaa 635 = Ala / Gly,Xaa 636 =Ser / Thr

[0084] SEQ ID NO: 373: Xaa 637 -Xaa 638 -Xaa 639 Xaa 637 =Arg / Lys / His,Xaa 638 =Val / Leu / Ile,Xaa 639 =Ser / Thr

[0085] SEQ ID NO: 374: Xaa 640 -Xaa 641 -Xaa 642 Xaa 640 =Leu / Val / Ile,Xaa 641 =Val / Leu / Ile,Xaa 642 =Ser / Thr

[0086] SEQ ID NO: 310: Xaa 94 -Xaa 95 -Xaa 96 -Xaa 97 -Xaa 98 -Xaa 99 -Xaa 100 -Xaa 101 -Xaa102 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 / Phe / Trp / Lys / Arg / His / Asp / Glu,Xaa 100 =Pro / Val / Leu / Ile,Xaa 101 =Trp / Phe / Tyr / Thr / Ser / Deletion,Xaa 102 =Thr / Ser / Lys / Arg / His

[0087] SEQ ID NO: 310 includes the following SEQ ID NO: 510. SEQ ID NO: 510: Xaa 783 -Xaa 784 -Xaa 785 -Xaa 786 -Xaa 787 -Xaa 788 -Xaa 789 -Xaa 790 -Xaa 791 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

[0088] The body 310 is also the same as the body 325 326 375 and 376 . 325: Yes 214 -No 215 -No 216 -No 217 -No 218 -No 219 -No 220 -No 221 -No 222 Yeah 214 =Gln / Asn,Xa 215 =Gln / Asn,Xa 216 =Cer / Thr,No 217 =Tyr / Phe / Trp,Xaa 218 =Asn / Gln,Xa 219 =Lion / Val / Dog,Dog 220 =Pro / Val / Lion / Home,Dog 221 =Trp / Phe / Tyr,Xaa 222 =Thr / Ser

[0089] 326: Yes 223 -No 224 -No 225 -No 226 -No 227 -No 228 -No 229 -No 230 -No 231 Yeah 223 =Lys / Arg / His, Rock 224 =Gln / Asn,Xa 225 =Cer / Thr,No 226 =Tyr / Phe / Trp,Xaa 227 =Thr / Ser,Knock 228 =Lion / Val / Dog,Dog 229 =Pro / Val / Lion / Home,Dog 230 =Trp / Phe / Tyr,Xaa 231 =Thr / Ser

[0090] 375: Yes643 -No 644 -No 645 -No 646 -No 647 -No 648 -No 649 -No 650 -No 651 Yeah 643 =Ser / Thr / His,No 644 =Asn / Gln,Xa 645 =Cer / Thr,No 646 =Cer / Thr,No 647 =Lys / Arg / His, Rock 648 =Val / Lion / Dog,Dog 649 =Pro,Yes 650 =Tyr / Phe / Trp,Xaa 651 =Ser / Thr

[0091] 376: Yes 652 -No 653 -No 654 -No 655 -No 656 -No 657 -No 658 -No 659 Yeah 652 =Asn / Gln,Xa 653 =Lys / Arg / His, Rock 654 =Val / Lion / Dog,Dog 655 =Lys / Arg / His, Rock 656 =Asp / Glu,Xa 657 =Val / Lion / Dog,Dog 658 =Cer / Thr,No 659 =Lys / Arg / His

[0092] (3) Remove the source(III) 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.

[0093] SEQ ID NO: 311: Xaa 102 -Xaa 103 -Xaa 104 -Xaa 105 -Xaa 106 -Xaa 107 -Xaa 108 -Xaa 109 -Xaa 110 -Xaa 111 -Xaa 112 -Xaa 113 -Xaa 114 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 / Tyr / Trp / Gly / Ala / deletion,Xaa 108 = Gln / Asn / Pro / deletion, Xaa 109 =Phe / Tyr / Trp / Val / Leu / Ile / deletion,Xaa 110 =Asp / Glu / Pro / Ala / Gly / Gln / Asn / Gly / Ala / Phe / Tyr / Trp / deletion,Xaa 111 = Ser / Thr / deletion, Xaa 112=Phe / Tyr / Trp / Ser / Thr / deletion,Xaa 113 =Arg / His / Lys / Phe / Tyr / Trp / Asp / Glu / deletion,Xaa 114 =Val / Leu / Ile / Phe / Tyr / Trp / deletion

[0094] SEQ ID NO: 311 includes SEQ ID NO: 511 below. SEQ ID NO:511: Xaa 792 -Xaa 793 -Xaa 794 -Xaa 795 -Xaa 796 -Xaa 797 -Xaa 798 -Xaa 799 -Xaa 800 -Xaa 801 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 / Ala / Gly,Xaa 801 =Phe / Tyr / Trp

[0095] As SEQ ID NO: 311, the following SEQ ID NOs: 327 to 329 and 377 are preferred. SEQ ID NO: 327: Xaa 232 -Xaa 233 -Xaa 234 -Xaa 235 -Xaa 236 -Xaa 237 -Xaa 238 -Xaa 239 -Xaa 240 Xaa 232 =Val / Leu / Ile,Xaa233 =Arg / Lys / His,stone 234 =His / Lys / Arg, Rock 235 =Gly / Ala,No 236 =Arg / His / Lys,stone 237 =Arg / His / Lys,stone 238 =Phe / Tyr / Trp,Xaa 239 =Asp / Glu,Xa 240 =Phe / Tyr / Trp

[0096] 328: Yes 241 -No 242 -No 243 -No 244 -No 245 -No 246 -No 247 -No 248 Yeah 241 =Ala / Gly,Rock 242 =Arg / Lys / His,stone 243 =His / Lys / Arg, Rock 244 =Arg / His / Lys,stone 245 =Cer / Thr,No 246 =Phe / Tyr / Trp,Xaa 247 =Pro / Ala / Gly,Dog 248 =Phe / Tyr / Trp

[0097] 329: Yes 249 -No 250 -No 251 -No 252 -No 253 -No 254 -No 255 -No 256 -No 257 -No 258 Yeah 249 =Ala / Gly,Rock 250 =Arg / Lys / His,stone 251 =His / Lys / Arg, Rock 252 =Gly / Ala,No 253=Arg / His / Lys,stone 254 =Cer / Thr,No 255 =Gln / Asn,Xa 256 =Phe / Tyr / Trp,Xaa 257 =Ala / Gly,Rock 258 =Phe / Tyr / Trp

[0098] 377: Yes 660 -No 661 -No 662 -No 663 -No 664 -No 665 -No 666 -No 667 -No 668 -No 669 -No 670 -No 671 -No 672 Yeah 660 =Cer / Thr,No 661 =Arg / Lys / His,stone 662 =Val / Lion / Dog,Dog 663 =Ala / Gly,Rock 664 =Ala / Gly,Rock 665 =Ala / Gly,Rock 666 =Pro,Yes 667 =Phe / Tyr / Trp,Xaa 668 =Phe / Tyr / Trp,Xaa 669 =Cer / Thr,No 670 =Phe / Tyr / Trp,Xaa 671 =Glu / Asp,Xa 672 =Phe / Tyr / Trp

[0099] 312: Yes 115 -No 116 -No 117 -No 118 -No 119 -No 120 -No 121 -No 122 -No 123 -No 124 -No125 -Xaa 126 Xaa 115 =Gln / Asn,Xaa 116 =Asn / Gln / Asp / Glu / Ser / Thr / deletion, Xaa 117 =Ser / Thr / Ile / Val / Leu / deletion,Xaa 118 = Ile / Val / Leu / deletion, Xaa 119 =Val / Leu / Ile / Gly / Ala / Tyr / Phe / Trp / Asp / Glu / deletion,Xaa 120 =His / Lys / Arg / Ser / Thr / Asn / Gln / deletion,Xaa 121 = Ser / Thr / deletion, Xaa 122 =Asp / Glu / Gly / Ala / Deletion, Xaa 123 =Gly / Ala / His / Lys / Arg / deletion,Xaa 124 =Asn / Gln / Asp / Glu / Lys / Arg / His / Deletion, Xaa 125 =Lys / Arg / His / Thr / Ser / deletion,Xaa 126 =Tyr / Phe / Trp

[0100] Examples of SEQ ID NO: 312 include SEQ ID NO: 512 below. SEQ ID NO:512: Xaa 802 -Xaa 803 -Xaa 804 -Xaa 805 -Xaa 806 -Xaa 807 -Xaa 808 -Xaa 809 -Xaa 810 -Xaa 811 -Xaa 812 -Xaa 813 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, Xaa808 =Ser / Thr / New, Yes 809 =Asp / Glu / CHD,Xa 810 =Gly / Ala / China,Daa 811 =Asn / Gln,Xa 812 =Lys / Arg / His / Thr / Ser,Xa 813 =Tyr / Phe / Trp

[0101] The 312 should also be the same as the 331 and the 378 should be the same. 330: Yes 258 -No 259 -No 260 -No 261 -No 262 -No 263 Yeah 258 =Gln / Asn,Xa 259 =Asn / Gln,Xa 260 =Home / Val / Lion,Dog 261 =Asn / Gln,Xa 262 =Lys / Arg / His, Rock 263 =Tyr / Phe / Trp

[0102] 331: Yes 264 -No 265 -No 266 -No 267 -No 268 -No 269 -No 270 -No 271 -No 272 -No 273 -No 274 Yeah 264 =Gln / Asn,Xa 265 =Cer / Thr,No 266 =Home / Val / Lion,Dog 267 =Val / Lion / Dog,Dog 268 =His / Lys / Arg, Rock 269 =Cer / Thr,No 270 =Asp / Glu,Xa 271= Gly / Ala, Xaa 272 =Asn / Gln,Xaa 273 =Thr / Ser,Xaa 274 =Tyr / Phe / Trp

[0103] SEQ ID NO: 378: Xaa 673 -Xaa 674 -Xaa 675 -Xaa 676 -Xaa 677 -Xaa 678 Xaa 673 =Gln / Asn,Xaa 674 =Glu / Asp,Xaa 675 =Val / Leu / Ile,Xaa 676 = Ala / Gly,Xaa 677 =Gln / Asn,Xaa 678 =Phe / Tyr / Trp

[0104] SEQ ID NO: 313: Xaa 127 -Xaa 128 -Xaa 129 Xaa 127 =Asn / Gln / Arg / Lys / His / Gly / Ala / Val / Leu / Ile,Xaa 128 =Thr / Ser / Val / Leu / Ile / His / Lys / Arg / Gly / Ala,Xaa 129 =Asn / Gln / Ser / Thr

[0105] Examples of SEQ ID NO: 313 include SEQ ID NO: 513 below. SEQ ID NO:513: Xaa 814 -Xaa 815 -Xaa 816 Xaa 814 =Asn / Gln / Arg / Lys / His,Xaa 815 =Thr / Ser / Val / Leu / Ile,Xaa 816 =Asn / Gln / Ser / Thr

[0106] As SEQ ID NO: 313, the following SEQ ID NOs: 332, 333 and 379 are preferred. 332: Yes 275 -No 276 -No 277 Yeah 275 =Asn / Gln,Xa 276 =Thr / Ser,Knock 277 =Asn / Gln

[0107] 333: Yes 278 -No 279 -No 280 Yeah 278 =Arg / Lys / His,stone 279 =Val / Lion / Dog,Dog 280 =Ser / Thr

[0108] 379: Yes 679 -No 680 -No 681 Yeah 679 =Ala / Gly,Rock 680 =Ala / Gly,Rock 681 =Ser / Thr

[0109] 314: Yes 130 -No 131 -No 132 -No 133 -No 134 -No 135 -No 136 -No 137 -No 138 Yeah 130 =Cys / Met / Phe / Tyr / Trp / Leu / Val / Ile,Xaa 131 =Gln / Asn,Xa 132 =His / Lys / Arg / Ser / Thr / Ala / Gly,Xaa 133 =Asn / Gln / Ala / Gly / Asp / Glu / His / Lys / Arg / Ser / Thr,Xaa 134 =Ser / Thr / His / Lys / Arg / Asn / Gln,Xaa 135=Trp / Phe / Tyr / Ala / Gly,Xaa 136 =Pro / Val / Leu / Ile,Xaa 137 =Val / Leu / Ile / Pro / Asp / Glu / Trp / Phe / Tyr,Xaa 138 =Thr / Ser

[0110] Examples of SEQ ID NO: 314 include SEQ ID NO: 514 below. 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 / Ser

[0111] As SEQ ID NO: 314, the following SEQ ID NOs: 334, 335 and 380 are preferred. SEQ ID NO: 334: 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,Xa 283 =His / Lys / Arg, Rock 284 =Asn / Gln,Xa 285 =Cer / Thr,No 286 =Trp / Phe / Tyr,Xaa 287 =Pro / Val / Lion / Home,Dog 288 =Val / Lion / Dog,Dog 289 =Thr / Ser

[0112] 335: Yes 290 -No 291 -No 292 -No 293 -No 294 -No 295 -No 296 -No 297 -No 298 Yeah 290 =Lion / Val / Dog,Dog 291 =Gln / Asn,Xa 292 =Cer / Thr,No 293 =Ala / Gly,Rock 294 =His / Lys / Arg, Rock 295 =Trp / Phe / Tyr,Xaa 296 =Pro / Val / Lion / Home,Dog 297 =Val / Lion / Dog,Dog 298 =Thr / Ser

[0113] 380: Yes 682 -No 683 -No 684 -No 685 -No 686 -No 687 -No 688 -No 689 -No 690 Yeah 682 =Val / Lion / Dog,Dog 683 =Gln / Asn,Xa 684 =His / Lys / Arg, Rock 685 =His / Lys / Arg, Rock686 =Gln / Asn,Xaa 687 =Trp / Phe / Tyr,Xaa 688 =Pro,Xaa 689 =Pro,Xaa 690 =Thr / Ser

[0114] Antibodies are glycoproteins produced by lymphocyte B cells that recognize and bind to specific proteins and other molecules. Antibodies bind specifically to specific molecules called antigens and cooperate with other biological molecules and cells to neutralize and eliminate antigen-bearing factors. Antibodies are essentially identical in molecular structure, consisting of a four-chain "Y" structure composed of two light chains and two heavy chain polypeptides. There are two types of light chains (L chains): lambda chains and kappa chains; all antibodies possess one of these. There are five types of heavy chains (H chains): gamma chains, mu chains, alpha chains, delta chains, and epsilon chains. The type of antibody (isotype) is determined by the difference in heavy chain structure. Antibodies are monomeric immunoglobulins composed of two heavy chains (gamma chains) and two light chains, each with two antigen-binding sites.

[0115] The lower half of the "Y" shape of an antibody, which corresponds to the vertical bar, is called the Fc region, and the upper half, the "V" shape, is called the Fab region. The Fc region has effector functions that trigger a reaction after the antibody binds to an antigen, while the Fab region has the function of binding to the antigen. The Fab and Fc regions of the heavy chain are connected by a hinge region, and the proteolytic enzyme papain found in papaya cleaves this hinge region, cleaving it into two Fab regions and one Fc region. The domain of the Fab region near the tip of the "Y" shape is called the variable region (V region) because its amino acid sequence varies widely, allowing it to bind to a variety of antigens. The variable region of the light chain is called the VL region, and the 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, and the constant region of the heavy chain is called the CH region, which is further divided into three parts: 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.

[0116] Each heavy chain and light chain contains a variable region consisting of approximately 100 or more amino acids, for example, 130 to 140 amino acids, that is primarily involved in antigen binding. The heavy chain variable region is also referred to as the heavy chain variable region (VH). The light chain variable region 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. VH and VL each contain three CDRs. That is, VH contains a first CDR (HCDR1), a second CDR (HCDR2), and a third CDR (HCDR3). VL contains a first CDR (LCDR1), a second CDR (LCDR2), and a third CDR (LCDR3). The regions other than the CDRs of the heavy and light chains have amino acid sequences that depend on, but are not limited to, the animal species and isotype.

[0117] The monoclonal antibody of the present invention can be produced by conventional methods. For example, first, an animal is immunized with an antigen. To produce an anti-A1 beta-casein antibody specific to A1 beta-casein, an antigen containing histidine at position 67 of A1 beta-casein is used. To produce 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. Laboratory animals that can be used include, for example, mice, rats, guinea pigs, rabbits, goats, sheep, donkeys, and chickens. The antigen can be administered, for example, by injection into the tail or intraperitoneally.

[0118] Next, B cells are extracted from the spleen or lymph nodes of the immunized animals and fused with myeloma cells to produce hybridomas. Hybridomas that produce antibodies with excellent specific binding ability to the antigen are screened from the produced hybridomas 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.

[0119] The binding activity of the monoclonal antibody according to 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 interferometry, or the like.

[0120] 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 to detect A1 beta-casein in particular. Specifically, a milk sample is mixed with the anti-A1 beta-casein antibody of the present invention, and A1 beta-casein is selectively bound to the milk sample for detection.

[0121] In the present disclosure, "detection" also includes "quantification," and for example, by preparing 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. Below, each step of the method for detecting A1 beta-casein according to the present invention will be explained, but the present invention is not limited to the following specific examples.

[0122] 1. Sample pre-treatment process In this step, the milk sample in which A1 beta-casein is to be detected is pretreated to improve detection sensitivity. This step is optional; for example, if the milk sample is to be directly subjected to measurement, this step is not necessary. Examples of pretreatment include diluting, defatting, and sterilizing the milk sample. The dilution ratio may be determined within a range that allows the concentration of A1 beta-casein to be determined from the ELISA measurement value using a calibration curve, and can be, for example, 1,000-fold or greater and 100,000-fold or less. PBS or the like can be used for dilution. Conventional defatting methods can be used. Sterilization can be either high-temperature sterilization or low-temperature sterilization.

[0123] 2.ELISA process In this step, A1 beta-casein contained in the milk sample is detected by ELISA (Enzyme-Linked Immunosorbent Assay). There are three types of ELISA known: direct, indirect, and sandwich. However, sandwich ELISA is described below for its high accuracy.

[0124] (1) Immobilization of capture antibodies In this step, 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, followed by incubation and washing.

[0125] The capture antibody may be an anti-A1 beta-casein antibody or an anti-A1 / A2 beta-casein antibody. According to the experimental findings of the present inventors, A1 beta-casein can be effectively detected whether the capture antibody is an anti-A1 beta-casein antibody or an anti-A1 / A2 beta-casein antibody.

[0126] (2) Mixing process of milk samples Next, the pretreated milk sample is added to the plate with the capture antibody immobilized on its surface, resulting in specific binding of A1 beta-casein, or A1 beta-casein and A2 beta-casein, contained in the milk sample to the antibody of the present invention. Afterwards, the plate is washed.

[0127] (3) Detection process Next, a detection antibody, an anti-A1 beta-casein antibody, is added to the plate after adding the milk sample, and the detection antibody is quantified by an enzymatic reaction. The epitope of the detection antibody is different from the epitope of the capture antibody. An enzyme-labeled capture antibody may be used, or a secondary antibody that specifically binds to the detection antibody and is enzyme-labeled may be used in addition to the detection antibody.

[0128] Quantification of the detection antibody can be performed using a method appropriate for the labeling enzyme conjugated to the detection antibody or secondary antibody. For example, if the labeling enzyme is horseradish peroxidase (HRP), hydrogen peroxide and a coloring agent are added. Examples of coloring agents used in combination with HRP 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). Absorbance at a wavelength appropriate for the coloring agent can be measured. Alternatively, if the labeling enzyme is alkaline phosphatase (ALP), nitrophenyl phosphate (pNPP) can be used as a coloring agent, and absorbance at 405 nm can be measured.

[0129] As mentioned above, if a calibration curve is created using a milk sample in which the amount and proportion of A1 beta-casein is known, it is possible to quantify the A1 beta-casein contained in the milk sample.

[0130] In addition, the proportion of Al beta-casein in A2 milk, which is considered to have a low proportion of Al beta-casein and is not harmful due to Al beta-casein, is said to be 30% by mass or less. According to the experimental findings of the present inventors, the monoclonal antibody of the present invention can sometimes detect Al beta-casein at 2% by mass or more of total beta-casein, making the Al beta-casein detection method of the present invention an excellent method for evaluating milk. However, the higher the proportion of Al beta-casein relative to total beta-casein, the easier it is to detect Al beta-casein. 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.

[0131] This application claims the benefit of priority based on Japanese Patent Application No. 2022-158177, filed on September 30, 2022. The entire contents of the specification of Japanese Patent Application No. 2022-158177, filed on September 30, 2022, are incorporated herein by reference. [Example]

[0132] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples, and it is possible to carry out the invention by making appropriate modifications within the scope of the above and below-described aims, and all such modifications are included in the technical scope of the present invention.

[0133] Example 1: Production of monoclonal antibodies by lymph node assay (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 μL) was mixed thoroughly and added to the upper syringe with a non-aqueous liquid pipette. The plunger of the lower syringe was pulled back to fill the interior with adjuvant, and then the plunger was returned to its original position, allowing the adjuvant to be returned to the upper syringe. The antigen proteins shown in Table 1 were dissolved in PBS to prepare 0.5 mg / mL solutions for mice and 1 mg / mL solutions for rats. Each solution (350 μL) 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 replaced. The plunger of the lower syringe was slowly pushed up to expel any air bubbles.

[0134] [Table 1]

[0135] The upper syringe was connected, and the syringe was held horizontally. The plunger was quickly moved back and forth approximately 10 times to emulsify the mixture. The resulting emulsion was drawn into one syringe, and the other syringe and needle connector were removed and a disposable needle was attached.

[0136] (2) Intramuscular injection into the base of the tail Mice or rats were anesthetized with sevoflurane and confirmed to be anesthetized by the lack of response to stimulation. 50 μL of the antigen-adjuvant emulsion was injected intramuscularly into the right side of the base of the tail (mice) or 100 μL (rats). The needle was withdrawn, a 5-second wait was given, and the same injection was then given to the left side. The injected mice or rats were placed in a cage at 37°C until they woke up, and then returned to their normal environment.

[0137] (3) Collection of iliac lymph nodes Approximately 14–21 days after injection, if the injection was successful, enlarged iliac lymph nodes were harvested. From six mice, 0.5–1.5 × 108 , and 0.5–2.0 × 10 from three rats. 8 In addition, with the electric cell fusion method, it is possible to expect the collection of 0.5 × 10 B cells. 8 The above cells are required. Specifically, immunized mice or rats were euthanized and their ventral skin was sprayed with 70% ethanol. The ventral skin and muscles were opened, avoiding the intestines, and enlarged iliac lymph nodes were located and collected. They were then transferred to Dulbecco's modified Eagle's medium (DMEM) and placed under a tissue culture hood.

[0138] (4) Isolation of B cells 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 through the wire mesh with a spatula. Using a pipette, cells (mainly B cells) were collected into a 50 mL tube. 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 procedure was repeated.

[0139] (5)Cell fusion Two days before fusion, SP2 / 0-Ag14 myeloma cells were split into DMEM using fresh culture medium to induce cells into logarithmic growth phase. The cells were centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed from the cell pellet. The cells were resuspended in DMEM (10 mL). The centrifugation and resuspension procedure was repeated. The number of myeloma cells was counted using a cell counting chamber and transferred to a new 50 mL tube at 2.0 × 10 7 A cell suspension was prepared. Also, count the number of B cells using a cell counting chamber and count 5.0 x 10 cells in a new 50 mL tube. 7 A cell suspension of 1000 μg was prepared and incubated at room temperature for 5 minutes. The B cell and myeloma cell suspensions were mixed and centrifuged at 1500 rpm for 5 minutes, the supernatant removed from the cell pellet, and the cells 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 removed from the cell pellet, and the cells resuspended in ECF buffer (1 mL). The suspensions of both cells (500 μL) were placed in the electrode chamber, and the chamber was placed in a cell fusion device ("Super Cell Fusion Device ECFG21" manufactured by Nepa Gene). The impedance was measured using an electric cell fusion generator, and a DC pulse voltage was set and a rectangular pulse was applied. The fusion procedure was repeated for the remaining cell suspension. The fused cell suspension was collected in a 50 mL tube containing HAT medium (40 mL). The fused cell suspension (100 μL) was seeded into a 96-well plate using a multichannel pipette and incubated at 37° C. in a CO 2 incubator.

[0140] (6) Hybridoma culture and cryopreservation Five days after cell fusion, the medium was carefully removed using an 8-channel aspirator, and HAT medium (200 μL) was added to each well of the plate. Eight days after cell fusion, the supernatant (100 μL) was used to identify wells that were positive for A1 beta-casein and negative for A2 beta-casein, or wells that were positive for A1 beta-casein and positive for A2 beta-casein, by ELISA. One day after ELISA confirmation, the cells in the wells were transferred to a 24-well plate containing HT medium (1 mL). After 2 to 3 days, 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 μL) was added, and the cells were suspended by pipetting. Storage medium (250 μL) was added, mixed well, transferred to a freezing tube, and stored at −80°C.

[0141] (7) Hybridoma cloning Frozen hybridoma cell stocks were removed from a -80°C freezer and rapidly thawed at 37°C. Once the cells were almost completely thawed, the vial containing the cells was placed in a tissue culture hood. After replacing the contents of the vial with HT medium, the number of viable hybridomas was counted using a cell counter and 2.0 × 10 cells were counted in 20 mL of HT medium in a 50 mL tube. 2 A suspension containing dispersed cells was prepared. The resulting hybridoma suspension (100 μL) was seeded into two 96-well plates and incubated at 37°C under CO2. Five days after seeding, HT medium (100 μL) was added, and eight days later, positive wells were confirmed by ELISA. One day after ELISA, positive hybridomas were transferred to a 24-well plate and subcultured for 2–3 days. Storage medium (250 μL) was added, mixed well, transferred to a cryotube, and stored at −80°C. To obtain large quantities of monoclonal antibodies, hybridomas from positive clones were thawed and cultured in 75 cm 2 The cells were grown until they reached the number required for 3 to 4 plates, and the culture supernatant containing the monoclonal antibody was collected during the logarithmic growth phase.

[0142] (8) Purification of monoclonal antibodies PBS (500 μL) was added to an empty column, and after allowing the PBS to drain, an approximately equal volume of affinity support for antibody purification (Protein G Sepharose 4 Fast Flow, GE Healthcare) was added after thorough mixing. The column was then washed once with 0.1 M glycine-HCl (pH 2.0, 2.5 mL) and twice with PBS (5 mL). 1 / 20 volume of 1M Tris (pH 8.0) was added to the culture supernatant of the positive clone, and a 15mL tube was placed under the column. To ensure binding of the antibody to Protein G, approximately half of the solution was added to the column, and after the solution had drained, the remaining half was added. The column was then washed three times with PBS (5mL). 500μL of 1M Tris (pH 8.0) was placed in a 5mL tube and placed under the column. 2.5mL of 0.1M glycine-HCl (pH 2.0) was added to drain the antibody, after which the mixture was immediately mixed well and transferred to a concentration tube. Centrifuged while monitoring the condition, and once the mixture had concentrated to approximately 500μL, the lower phase was discarded and approximately 5mL of PBS was added to the upper phase. After thorough mixing by inversion, the mixture was further centrifuged to concentrate to approximately 300μL, transferred to a 1.5mL tube, and the total volume was adjusted to approximately 500μL with PSB. The antibody concentration was confirmed by electrophoresis.

[0143] (9) Amino acid sequence of monoclonal antibody Total RNA was extracted from the positive clones, and cDNA was prepared from the total RNA using a cDNA preparation reagent composition (SuperScript III First-Strand Synthesis SuperMix, Thermo Fisher Scientific). The CDR gene was amplified by reverse transcription PCR using a high-speed PCR enzyme (KOD-Plus-Neo, Toyobo) and primers corresponding to the antibody's antigen complementarity-determining region (CDR). The nucleotide sequence and the amino acid sequence of the CDR were determined by outsourcing. The amino acid sequences of the CDRs of rat-derived anti-A1 beta-casein antibodies are shown in Table 2, those of rat-derived anti-A1 / A2 beta-casein antibodies are shown in Table 3, and those of mouse-derived anti-A1 beta-casein antibodies are shown in Table 4.

[0144] [Table 2]

[0145] [Table 3]

[0146] [Table 4]

[0147] 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 times and mixed to obtain the composition shown in Table 5 to prepare A1 / A2 beta-casein skim milk.

[0148] [Table 5]

[0149] The #27-1 anti-A1 / A2 beta-casein antibody used as the capture antibody was diluted with PBS to prepare a 1.5 μg / 100 μL solution, which was then added to a 96-well plate at 100 μL / well. The plate was then covered and incubated overnight at 37°C. The liquid was then discarded, washed twice with PBS (200 μL), and 1% BSA / PBS (200 μL) was added. The plate was then covered and incubated for 1 hour at 37°C or overnight at 4°C. The liquid was then discarded, washed twice with PBS (200 μL), and 100 μL of a milk sample diluted 100-fold with PBS was added. The plate was then covered and incubated for 1.5 to 2 hours at 37°C. The liquid was then discarded and washed three times with PBS (200 μL). Next, the #6-3 anti-A1 beta-casein antibody was diluted with 1% BSA / PBS to prepare a 1.5 μg / 100 μL solution, and the resulting solution (100 μL) was added to the plate. 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 μL). The HRP-labeled secondary antibody was diluted 20,000-fold with 1% BSA / PBS, and 100 μL of the diluted solution was added. The plate was covered and incubated at room temperature for 1 hour. The solution was discarded and washed three times with 200 μL of PBS. A separate 0.4 mg / mL o-phenylenediamine dihydrochloride + 0.012% hydrogen peroxide solution in PBS was added in 150 μL portions and allowed to stand at room temperature for 30–60 minutes. The reaction was then stopped by adding 50 μL of 3 M sulfuric acid, and the absorbance was measured at 492 nm. Measurements were performed three times on the same day, and the average optical density (OD) values ​​were calculated. The results are shown in Figure 1. In Figures 1–3, the horizontal axis indicates the amount of A1 beta-casein per μL of undiluted milk sample. As shown in Figure 1, the relationship between the A1 beta-casein content and the measured OD value was y=-8×10 -5 x 2 The approximate equation is +0.0138x+0.1229, and its coefficient of determination R 2 The OD value was very high at 0.998. Furthermore, despite the fact that the presence of A2 beta-casein inhibits the measurement of A1 beta-casein, when a sample contained relatively high amounts of A2 beta-casein, specifically when the A1:A2 ratio was 1:30-50, the OD value measured showed a significant difference compared to the measured OD value of a sample that did not contain A1 beta-casein. Generally, A2 milk, in which the influence of A1 beta-casein is reduced, refers to milk in which the proportion of A1 beta-casein in the total beta-casein is suppressed to 30% or less. Therefore, according to the present invention, it has been demonstrated that the beta-casein contained in milk can be adequately evaluated. Furthermore, based on the beta-casein concentration in the measurement sample, the actual milk to be measured can be skimmed and diluted 10,000 times and subjected to ELISA measurement under the above conditions.

[0150] Example 3: Quantification of A1 beta-casein by sandwich ELISA ELISA measurements were 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 using anti-A1 beta-casein antibody were similar to those obtained using anti-A1 / A2 beta-casein antibody. Specifically, the correlation between the A1 beta-casein content and the measured OD value was y = -7 × 10 -5 x 2 The approximate equation is +0.0135x+0.1273, and its coefficient of determination R 2 The result was a very high 0.9996. Furthermore, a curve very similar to that in Example 2 was obtained, demonstrating that the combination of #17-2 anti-A1 beta-casein antibody and #6-3 anti-A1 beta-casein antibody also enables detection of A1 beta-casein in milk feed containing A2 beta-casein.

[0151] Example 4: Quantification of A1 beta-casein by sandwich ELISA ELISA measurements were 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. As shown in Figure 3, the results were similar to those of Examples 2 and 3. Specifically, the relationship between the A1 beta-casein content and the measured OD value was y = -6 x 10 -5 x 2 The approximate equation is +0.012x+0.1312, and its coefficient of determination R 2 The value was very high at 0.9939. Also, a curve very similar to that in Examples 2 and 3 was obtained. However, in Examples 2 and 3, in the milk samples containing 200 ng / μL or more of A1 beta-casein before dilution, the value was 2 × 10 -2 In this example, A1 beta-casein was detectable in milk samples containing 1000 ng / μL or more of A1 beta-casein before dilution, whereas the measured OD values ​​showed that in milk samples containing 1000 ng / μL or more of A1 beta-casein before dilution, the level was 1 × 10 -1 A1 beta-casein was detectable in milk samples containing ng / μL or more of A1 beta-casein.

[0152] 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 Figure 4.

[0153] [Table 6]

[0154] As shown in Table 6 and Figure 4, the relationship between the A1 beta-casein content and the measured OD value was y = -3.1 × 10 -3 x 2 The approximate equation is +0.1339x+0.1847, and its coefficient of determination R 2 The value was very high at 0.9998. It was also revealed that measurement is possible not only for skim milk but also for raw milk. Furthermore, it was shown that the present invention can detect A1 beta-casein in raw milk as long as the mass ratio to A2 beta-casein is 1 / 200 or more.

[0155] 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 Figure 5.

[0156] [Table 7]

[0157] As shown in Table 7 and Figure 5, the relationship between the A1 beta-casein content and the measured OD value was y = -2.0 × 10 -4 x 2 The approximate equation is +0.025x+0.1696, and its coefficient of determination R 2 The value was very high at 0.9653. It was also revealed that measurement is possible not only for skim milk but also for raw milk. Furthermore, it was shown that the present invention can detect A1 beta-casein in raw milk as long as the mass ratio of A1 beta-casein to A2 beta-casein is 1 / 100 or more.

[0158] Example 7 We have produced three antibodies with the following CDR amino acid sequences: 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). Each antibody has excellent selective binding ability to A1 beta-casein or both A1 and A2 beta-casein.

[0159] [Table 8]

[0160] [Table 9]

[0161] [Table 10-1]

[0162] [Table 10-2]

Claims

1. (1) a heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 10, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 17; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 25, a light chain complementarity determining region 2 comprising the amino acid sequence of Asn-Ala-Asn, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 31; (2) a heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 5, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 11, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 18; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 26, a light chain complementarity determining region 2 comprising the amino acid sequence of Asp-Thr-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 32; (3) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 11, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 19; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 27, a light chain complementarity determining region 2 comprising the amino acid sequence of Asp-Thr-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 33; (4) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 73, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 115; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 164, a light chain complementarity determining region 2 comprising the amino acid sequence of Leu-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 193; (5) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 74, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 116; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 191, a light chain complementarity determining region 2 comprising the amino acid sequence of Leu-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 194; (6) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 10, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 117; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 165, a light chain complementarity determining region 2 comprising the amino acid sequence of Ser-Gly-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 195; (7) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 39, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 111, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 118; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 25, a light chain complementarity determining region 2 comprising the amino acid sequence of Asn-Ala-Asn, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 196; (8) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 40, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 75, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 119; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 28, a light chain complementarity determining region 2 comprising the amino acid sequence of Asn-Pro-Asn, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 197; (9) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 41, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 76, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 120; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 26, a light chain complementarity determining region 2 comprising the amino acid sequence of Asp-Thr-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 33; (10) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 42, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 10, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 18; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 26, a light chain complementarity determining region 2 comprising the amino acid sequence of Asp-Thr-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 198; (11) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 10, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 18; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 26, a light chain complementarity determining region 2 comprising the amino acid sequence of Asp-Thr-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 32; (12) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 75, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 18; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 26, a light chain complementarity determining region 2 comprising the amino acid sequence of Glu-Ile-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 32; (13) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 77, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 121; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 166, a light chain complementarity determining region 2 comprising the amino acid sequence of Asp-Thr-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 199; (14) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 78, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 122; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 165, a light chain complementarity determining region 2 comprising the amino acid sequence of Ser-Gly-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 195; (15) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 79, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 122; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 165, a light chain complementarity determining region 2 comprising the amino acid sequence of Ser-Gly-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 195; (16) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 43, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 80, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 123; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 167, a light chain complementarity determining region 2 comprising the amino acid sequence of Trp-Met-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 200; or (17) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 44, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 81, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 124; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 168, a light chain complementarity determining region 2 comprising the amino acid sequence of Asn-Ala-Asn, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 201; (18) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 44, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 81, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 124; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 168, a light chain complementarity determining region 2 comprising the amino acid sequence of Asn-Ala-Asn, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 201; (19) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 44, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 81, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 124; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 168, a light chain complementarity determining region 2 comprising the amino acid sequence of Tyr-Ala-Asn, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 201; (20) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 45, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 82, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 125; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 168, a light chain complementarity determining region 2 comprising the amino acid sequence of Tyr-Ala-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 201; (21) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 44, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 83, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 125; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 168, a light chain complementarity determining region 2 comprising the amino acid sequence of Tyr-Ala-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 202; (22) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 46, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 84, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 126; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 169, a light chain complementarity determining region 2 comprising the amino acid sequence of Leu-Ala-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 203; (23) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 46, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 85, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 126; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 169, a light chain complementarity determining region 2 comprising the amino acid sequence of Leu-Ala-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 203; (24) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 47, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 86, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 127; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 170, a light chain complementarity determining region 2 comprising the amino acid sequence of Gln-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 204; (25) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 47, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 87, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 127; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 170, a light chain complementarity determining region 2 comprising the amino acid sequence of Gln-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 204; (26) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 48, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 88, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 127; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 170, a light chain complementarity determining region 2 comprising the amino acid sequence of Gln-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 204; or (27) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 49, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 89, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 128; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 170, a light chain complementarity determining region 2 comprising the amino acid sequence of Gln-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 204; A monoclonal antibody characterized by binding to A1 beta-casein.

2. (1) a heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 8, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 14, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 23; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 30, a light chain complementarity determining region 2 comprising the amino acid sequence of Trp-Ala-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 37; (2) a heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 9, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 15, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 24; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 30, a light chain complementarity determining region 2 comprising the amino acid sequence of Trp-Ala-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 38; (3) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 9, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 16, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 23; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 30, a light chain complementarity determining region 2 comprising the amino acid sequence of Trp-Ala-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 37; (4) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 69, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 112, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 159; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 190, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 224; or (5) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 8, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 16, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 140; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 30, a light chain complementarity determining region 2 comprising the amino acid sequence of Trp-Ala-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 37; (6) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 9, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 15, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 23; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 30, a light chain complementarity determining region 2 comprising the amino acid sequence of Trp-Ala-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 212; (7) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 9, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 97, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 141; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 30, a light chain complementarity determining region 2 comprising the amino acid sequence of Trp-Ala-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 213; (8) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 56, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 98, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 142; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 30, a light chain complementarity determining region 2 comprising the amino acid sequence of Trp-Ala-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 212; (9) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 57, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 15, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 143; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 176, a light chain complementarity determining region 2 comprising the amino acid sequence of Trp-Ala-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 212; or (10) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 58, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 15, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 144; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 30, a light chain complementarity determining region 2 comprising the amino acid sequence of Trp-Ala-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 212; (11) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 59, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 99, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 145; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 177, a light chain complementarity determining region 2 comprising the amino acid sequence of Trp-Ala-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 212; or (12) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 60, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 100, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 146; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 178, a light chain complementarity determining region 2 comprising the amino acid sequence of Trp-Ala-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 214; or (13) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 60, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 101, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 146; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 179, a light chain complementarity determining region 2 comprising the amino acid sequence of Trp-Ala-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 214; or (14) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 61, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 102, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 147; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 180, a light chain complementarity determining region 2 comprising the amino acid sequence of Trp-Ala-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 215; (15) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 62, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 103, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 148; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 181, a light chain complementarity determining region 2 comprising the amino acid sequence of Trp-Ala-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 216; (16) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 62, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 103, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 149; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 180, a light chain complementarity determining region 2 comprising the amino acid sequence of Trp-Ala-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 215; (17) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 63, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 104, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 150; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 182, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 217; (18) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 64, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 105, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 151; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 183, a light chain complementarity determining region 2 comprising the amino acid sequence of Ala-Thr-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 218; (19) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 65, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 106, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 152; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 184, a light chain complementarity determining region 2 comprising the amino acid sequence of Leu-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 219; (20) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 65, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 105, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 153; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 184, a light chain complementarity determining region 2 comprising the amino acid sequence of Leu-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 219; (21) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 66, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 105, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 154; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 184, a light chain complementarity determining region 2 comprising the amino acid sequence of Leu-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 219; (22) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 65, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 105, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 155; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 184, a light chain complementarity determining region 2 comprising the amino acid sequence of Leu-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 219; (23) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 65, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 105, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 156; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 184, a light chain complementarity determining region 2 comprising the amino acid sequence of Leu-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 219; (24) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 67, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 107, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 157; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 184, a light chain complementarity determining region 2 comprising the amino acid sequence of Leu-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 219; (25) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 68, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 105, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 158; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 185, a light chain complementarity determining region 2 comprising the amino acid sequence of Leu-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 220; or (26) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 65, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 105, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 159; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 186, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 221; or (27) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 65, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 105, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 159; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 186, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 222; or (28) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 65, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 105, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 159; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 187, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 223; or (29) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 65, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 108, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 159; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 187, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 223; or (30) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 65, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 109, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 159; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 187, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 223; or (31) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 65, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 110, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 159; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 188, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 223; or (32) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 65, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 111, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 159; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 186, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 221; or (33) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 65, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 111, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 159; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 189, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 221; (34) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 66, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 105, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 159; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 186, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 221; or (35) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 66, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 109, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 159; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 190, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 224; or (36) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 65, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 113, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 159; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 186, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 221; or (37) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 70, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 113, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 159; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 191, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 221; (38) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 70, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 114, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 159; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 191, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 221; (39) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 71, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 114, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 159; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 186, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 221; or (40) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 68, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 105, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 159; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 186, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 221; or (41) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 65, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 114, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 160; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 186, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 221; or (42) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 72, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 105, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 160; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 186, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 225; or (43) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 65, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 105, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 161; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 186, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 226; (44) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 65, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 105, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 161; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 186, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 227; or (45) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 65, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 105, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 162; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 186, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 227; or (46) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 65, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 105, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 163; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 186, a light chain complementarity determining region 2 comprising the amino acid sequence Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 227; or (47) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 65, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 105, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 161; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 192, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 226; A monoclonal antibody characterized by binding to A1 beta-casein.

3. The monoclonal antibody according to claim 1 or 2, further comprising an Fc region.

4. A hybridoma comprising nucleic acids encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody of claim 1 or 2.

5. 1. A method for detecting A1 beta-casein in a milk sample, comprising: A method comprising the step of mixing the milk sample with the monoclonal antibody of claim 1 or 2.

6. Further, a step of selecting a detection antibody from the monoclonal antibodies of claim 1 or 2, and selecting a capture antibody having an epitope different from that of the detection antibody from the monoclonal antibodies of claim 1 or 2 or the following monoclonal antibodies comprising heavy chain variable regions and light chain variable regions and binding to A1 beta-casein and A2 beta-casein: (1) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 12, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 20; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 28, a light chain complementarity determining region 2 comprising the amino acid sequence of Asn-Thr-Asn, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 34; (2) a heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 12, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 21; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 28, a light chain complementarity determining region 2 comprising the amino acid sequence of Asn-Thr-Asn, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 35; (3) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 13, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 22; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 29, a light chain complementarity determining region 2 comprising the amino acid sequence of Arg-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 36; (4) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 13, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 22; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 29, a light chain complementarity determining region 2 comprising the amino acid sequence of Arg-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 36; (5) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 12, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 129; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 28, a light chain complementarity determining region 2 comprising the amino acid sequence of Asn-Lys-Asn, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 35; (6) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 90, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 130; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 28, a light chain complementarity determining region 2 comprising the amino acid sequence of Asn-Thr-Asn, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 205; (7) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 50, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 12, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 131; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 28, a light chain complementarity determining region 2 comprising the amino acid sequence of Asn-Thr-Asn, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 206; (8) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 51, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 91, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 132; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 171, a light chain complementarity determining region 2 comprising the amino acid sequence of Asn-Ser-Asn, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 207; (9) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 52, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 92, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 133; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 171, a light chain complementarity determining region 2 comprising the amino acid sequence of Lys-Ala-Asn, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 207; (10) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 12, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 134; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 28, a light chain complementarity determining region 2 comprising the amino acid sequence of Asn-Thr-Asn, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 35; (11) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 90, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 135; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 172, a light chain complementarity determining region 2 comprising the amino acid sequence of Asn-Thr-Asn, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 35; (12) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 53, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 93, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 136; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 173, a light chain complementarity determining region 2 comprising the amino acid sequence of Gly-Ala-Thr, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 208; (13) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 54, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 94, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 137; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 174, a light chain complementarity determining region 2 comprising the amino acid sequence of Gln-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 209; (14) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 55, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 95, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 138; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 170, a light chain complementarity determining region 2 comprising the amino acid sequence of Gln-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 210; or (15) A heavy chain variable region comprising a heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 60, a heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 96, and a heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 139; and a light chain variable region comprising a light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 175, a light chain complementarity determining region 2 comprising the amino acid sequence of Leu-Val-Ser, and a light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO: 211; Immobilizing the capture antibody on a plate; and 6. The method of claim 5, further comprising the step of mixing the detection antibody with the mixture of the capture antibody and the milk sample.

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