Antibody Combination Against Regenerating Islet-Derived Protein 1a And Detection Kit Comprising Same

Abstract

The present invention provides an antibody combination that can be used to detect regenerating islet-derived protein 1? (REGIA), each antibody in the antibody combination can specifically bind to REGIA with high affinity so as to form a double-antibody sandwich form, thereby enabling qualitative and quantitative detection of human REGIA. The antibody combination or an ELISA detection kit comprising the antibody combination can be used for auxiliary diagnosis or disease risk prediction of REGIA-related diseases.

Claims

1. An antibody combination, and the antibody combination comprising: (i) Antibody 1: The Antibody 1 consists of heavy chains (HC) and light chains (LC), wherein the heavy chains include a heavy chain CDR1 (H-CDR1) illustrated in SEQ ID NO. 1, a heavy chain CDR2 (H-CDR2) illustrated in SEQ ID NO. 2 and a heavy chain CDR3 (H-CDR3) illustrated in SEQ ID NO. 3 and the light chains include a light chain CDR1 (L-CDR1) illustrated in SEQ ID NO. 6, a light chain CDR2 (L-CDR2) illustrated in SEQ ID NO. 7 and a light chain CDR3 (L-CDR3) illustrated in SEQ ID NO. 8; and (ii) Antibody 2: The Antibody 2 consists of heavy chains (HC) and light chains (LC), wherein the heavy chains include a heavy chain CDR1 (H-CDR1) illustrated in SEQ ID NO. 11, a heavy chain CDR2 (H-CDR2) illustrated in SEQ ID NO. 12 and a heavy chain CDR3 (H-CDR3) illustrated in SEQ ID NO. 13 and the light chains include a light chain CDR1 (L-CDR1) illustrated in SEQ ID NO. 16, a light chain CDR2 (L-CDR2) illustrated in SEQ ID NO. 17 and a light chain CDR3 (L-CDR3) illustrated in SEQ ID NO. 18.

2. An antibody combination according to claim 1, which is characterized in that the Antibody 1 and the Antibody 2 are the binding agents that can specifically bind to the regenerating islet-derived protein 1? (REG1A).

3. An antibody combination according to claim 1, which is characterized in that the heavy chains of the Antibody 1 include a heavy chain variable region (VH), the light chains include a light chain variable region (VL), and the VH of the Antibody 1 includes an amino acid sequence illustrated in SEQ ID NO. 4 and the VL includes an amino acid sequence illustrated in SEQ ID NO. 9; and The heavy chains of the Antibody 2 include a heavy chain variable region (VH), the light chains include a light chain variable region (VL), and the VH of the Antibody 2 includes an amino acid sequence illustrated in SEQ ID NO. 14 and the VL includes an amino acid sequence illustrated in SEQ ID NO. 19.

4. An antibody combination according to claim 3, which is characterized in that the Antibody 1 and the Antibody 2 are selected from mouse, chimeric and humanized antibodies.

5. An antibody combination according to claim 3, which is characterized in that the Antibody 1 and the Antibody 2 are selected from monoclonal, Fab, Fv and ScFv antibodies.

6. An antibody combination according to claim 5, which is characterized in that each of the Antibody 1 and the Antibody 2 is a monoclonal antibody, consisting of 2 heavy chains and 2 light chains.

7. An antibody combination according to claim 6, which is characterized in that the Antibody 1 and the Antibody 2 are the mouse IgG1/Kappa isotypes.

8. An antibody combination according to claim 6, which is characterized in that the heavy chains of the Antibody 1 include an amino acid sequence illustrated in SEQ ID NO. 5 and the light chains include an amino acid sequence illustrated in SEQ ID NO. 10; and, the heavy chains of the Antibody 2 include an amino acid sequence illustrated in SEQ ID NO. 15 and the light chains include an amino acid sequence illustrated in SEQ ID NO. 20.

9. An antibody combination according to claim 1, which is characterized in that the Antibody 1 or the Antibody 2 has a detectable label.

10. An antibody combination according to claim 9, which is characterized in that the Antibody 2 has a detectable label.

11. An antibody combination according to claim 10, which is characterized in that the detectable label is an enzyme label, radioactive label, luminescent label, color rendering label, hapten, metal complex or metal.

12. A use of the antibody combination to prepare a reagent for detecting the regenerating islet-derived protein 1? (REG1A) according to claim 1.

13. A use of the antibody combination to prepare a reagent for auxiliary diagnosis or disease risk prediction of REG1A-related diseases according to claim 1.

14. A use according to claim 12, which is characterized in that the regenerating islet-derived protein 1? (REG1A) is a human regenerating islet-derived protein 1a.

15. A use according to claim 13, which is characterized in that REG1A expression rises in the REG1A-related diseases.

16. A use according to claim 15, which is characterized in that the REG1A-related diseases are diseases of the digestive system.

17. A use according to claim 16, which is characterized in that the diseases of the digestive system are diseases of the digestive tract.

18. A use according to claim 17, which is characterized in that the disease of the digestive tract is IBD, peptic ulcer, adenomatous polyp, Stage 1, Stage 2, Stage 3 or Stage 4 colorectal cancer, gastroenteritis or gastric cancer.

19. A use according to claim 16, which is characterized in that the disease of the digestive system is pancreatitis.

20. A use according to claim 19, which is characterized in that the pancreatitis is acute pancreatitis.

21. A use according to claim 12, which is characterized in that the reagent can be used in the following detection methods: chemiluminescence immunoassay, immunonephelometry, enzyme-linked immunosorbent assay (ELISA), Western blotting, antibody microarray, immunoprecipitation, and radioimmunoassay (RIA).

22. A use according to claim 21, which is characterized in that the reagent is a detection reagent used for ELISA.

23. A use according to claim 22, which is characterized in that the detection reagent is a detection reagent used for DAS-ELISA; wherein the ELISA is used to detect the regenerating islet-derived protein 1? (REG1A) in whole blood, serum or plasma.

24. A use according to claim 23, which is characterized in that, in the ELISA, the Antibody 1 is used as a capture antibody and the Antibody 2 is used as a detection antibody.

25. A kit that includes the antibody combination according to claim 1.

26. A kit according to claim 25, which is characterized in that the kit is a kit used in the following detection methods: chemiluminescence immunoassay, immunonephelometry, enzyme-linked immunosorbent assay (ELISA), Western blotting, antibody microarray, immunoprecipitation, and radioimmunoassay (RIA).

27. A kit according to claim 26, which is characterized in that the kit is an ELISA kit.

28. A kit according to claim 27, which is characterized in that the kit is a DAS-ELISA kit.

29. A kit according to claim 28, which is characterized in that the kit includes the Antibody 1 that is used as a capture antibody and the Antibody 2 that is used as a detection antibody.

30. A kit according to claim 29, which is characterized in that the Antibody 2 has a detectable label.

31. A kit according to claim 29, which is characterized in that the detectable label is an enzyme label, radioactive label, luminescent label, color rendering label, hapten, metal complex or metal.

32. A kit according to claim 25, which is characterized in that the kit also includes other reagents necessary for using ELISA to detect the existence or concentration of REG1A in biological samples.

33. A kit according to claim 32, which is characterized in that the other reagents are one or more of the following items: REG1A calibrator and/or control material, antibody diluent, wash buffer, blocking buffer, diluent of the detected sample, ELISA plate, color-developing agent and stop buffer.

34. A method for the auxiliary diagnosis or disease risk prediction of REG1A-related diseases, including: using an antibody combination according to claim 1.

35. A method according to claim 34, which is characterized in that the method also includes: comparing the detected concentration of REG1A with a reference level, and a higher concentration of REG1A in the biological samples indicates that the subjects suffer from a REG1A-related disease or are at risk of suffering from a REG1A-related disease.

36. A method for the auxiliary diagnosis or disease risk prediction of REG1A-related diseases, including: using a kit according to claim 25 to detect the concentration of REG1A in biological samples from subjects.

Description

DESCRIPTION OF FIGURES

[0046] Combined with drawings, the embodiments provided in the present invention are explained in detail hereunder, wherein:

[0047] FIG. 1 is a schematic diagram of the structure of an antibody in the combination provided in the present invention when the antibody is a monoclonal antibody.

[0048] FIG. 2 illustrates the results of antibody pairing and screening.

[0049] FIG. 3 illustrates the results of antibody pairing and screening.

[0050] FIG. 4 is a ROC curve drawn according to the detection results of REG1A from different serum samples, wherein: [0051] 4-1: patients with Stage 1 colorectal cancer; 4-2: patients with Stage 2 colorectal cancer; 4-3: patients with Stage 3 colorectal cancer; 4-4: patients with Stage 4 colorectal cancer; 4-5: all patients with colorectal cancer; 4-6: patients with gastric cancer; 4-7: patients with gastroenteritis; 4-8: patients with adenomatous polyp; 4-9: patients with IBD; 4-10: patients with peptic ulcer; 4-11: patients with acute pancreatitis.

[0052] FIG. 5 illustrates the concentrations of REG1A in serum before and after the effective treatment of 29 patients with active IBD (****, P<0.001).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] The present invention is described by reference to the preferred embodiments. Those skilled in the art can understand that these embodiments are intended to illustrate the present invention and not to limit the range of the present invention in any way.

[0054] The experiment methods in the following embodiments are conventional methods unless otherwise specified. Raw materials and reagent materials used in the following embodiments are commercially available off-the-shelf (COTS) items unless otherwise specified.

[0055] The recombinant human REG1A is recombined and prepared according to NCBI reference sequence (NP_002900.2).

Embodiment 1 Acquisition of a Monoclonal Antibody for the Human Regenerating Islet-Derived Protein 1? (REG1A)

[0056] Immunization: Mice are immunized by giving them a multipoint subcutaneous injection of a recombinant human REG1A, and this step is repeated for 3-4 times. After immunization, a small amount of blood is taken from these mice for potency assay and the spleens of mice with high potency are selected for further fusion.

[0057] Cell fusion: Splenocytes from mice are selected, grinded, and screened through a cell strainer, and then are centrifuged and washed with a culture solution. In this way, splenic lymphocyte suspension is obtained. After that, myeloma cells and splenocytes are mixed proportionally, and a fusion agent is added to the mixture for fusion.

[0058] Screening: A HAT selection culture solution is used for culturing, to screen out hybridoma cells, and meanwhile, the supernatant is measured and cultured with ELISA, to screen out the hybridoma cells producing antibodies that can be used to specifically recognize the human REG1A. Then, the screened positive hybridoma cells are cloned with the limiting dilution method to get stable monoclonal hybridoma cells.

[0059] Antibody production: The selected hybridoma cells are taken for expanded culture, the cultured supernatant is taken and purified with affinity chromatography to get monoclonal antibodies.

[0060] A total of 5 monoclonal antibodies are obtained and are mouse IgG1/Kappa isotypes after examination, which are separately named in the present invention: antibody 4H8F1; antibody 12F12A10; antibody 11D11B11; antibody 21B11D2; and antibody 25F3C4. The antibody sequence is as follows:

(i) 4H8F1:

[0061] Heavy chain (SEQ ID NO. 5 (removing signal peptide); wherein, the heavy chain variable region is SEQ ID NO. 4; CDRs are SEQ ID NO. 1/SEQ ID NO. 2/SEQ ID NO. 3 in order):

TABLE-US-00001 MGRLTSSFLLLIVPAYVLSQVTLKESGPGILQPSQTLSLTCSFSG FSLSTSGMSVGWIRQPSGKGLEWLAHIWWNDDKFYNPALKSRLTI SKDTSKNQIFLKIASVVTADSATYYCARIEEGWFAYWGQGTLVTV STAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNS GSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPA SSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLT PKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFR SVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAP QVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENY KNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHH TEKSLSHSPGK

[0062] Light chain (SEQ ID NO. 10 (removing signal peptide); wherein, the light chain variable region is SEQ ID NO. 9, CDRs are SEQ ID NO. 6/SEQ ID NO. 7/SEQ ID NO. 8 in order):

TABLE-US-00002 MKSQTQVFVFLLLCVSAAHGSIVMTQTPKFLLVSVGDRVTITCKA SQTMSNDVAWYQQKPGQSPKLLIYYASNRYTGVPDRFTGSGYGTD FTFTISTVQAEDLAVYFCQQDYSSPLTFGAGTKLELKRADAAPTV SIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVL NSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPI VKSFNRNEC

(ii) 12F12A10:

[0063] Heavy chain (SEQ ID NO. 15 (removing signal peptide); wherein, the heavy chain variable region is SEQ ID NO. 14; CDRs are SEQ ID NO. 11/SEQ ID NO. 12/SEQ ID NO. 13 in order):

TABLE-US-00003 MERHWIFLFLLSITAGVHSQVQLQQSATELARPGASVKMSCKASG YTFTSYMMHWVKQRPGQGLEWIGYINPSSGYTDYNQKFKDKTTLT ADKSSSTAYMQLSSLTSEDSAVYYCARYRYPHYFDYWGQGTTLTV SSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNS GSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPA SSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLT PKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFR SVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAP QVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENY KNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHH TEKSLSHSPGK

[0064] Light chain (SEQ ID NO. 20 (removing signal peptide); wherein, the light chain variable region is SEQ ID NO. 19; CDRs are SEQ ID NO. 16/SEQ ID NO. 17/SEQ ID NO. 18 in order):

TABLE-US-00004 MDFQVQIFSFLLISASVIISRGQIVLTQSPAIMSASPGEKVTMTC SASSSVSYIHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGT SYSLTISSMEAEDAAIYYCQQWSSNPPTFGAGTKLELKRADAAPT VSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGV LNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSP IVKSFNRNEC
(iii) Antibody 11D11B11: Heavy chain (SEQ ID NO. 21 (removing signal peptide)):

TABLE-US-00005 MSSEHRPLTMNFGLRLIFLVLTLKGVQCDVKLVESGGGLVKPGGS LKLSCAASGFTFSSFSMSWFRQTPDKRLEWVATISSGGSSTYYPD SVKGRFTISRDNAKNTLYLQMTSLKSEDTAIFYCRGGYYGNYDPL DYWGQGTSVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGY FPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPS QSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPS VFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEV HTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLP APIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMP EDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVE RNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0065] Light chain (SEQ ID NO. 22 (removing signal peptide)):

TABLE-US-00006 MGIKMESQTQVFVYMLLWLSGVDGDIVMIQSQKFMSTSVGDRVSI TCTASHNVDSNVAWYQQKPGQSPQALIYSASYRYSGVPDRFTGSG SGTDFTLTINNVQSEDLAEYFCQQYNSYPLTFGGGTRLEIKRADA APTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQ NGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATH KTSTSPIVKSFNRNEC

(iv) Antibody 21B11D2:

[0066] Heavy chain (SEQ ID NO. 23 (removing signal peptide)):

TABLE-US-00007 MEWSWVFLFLLSVIAGVQSQAQLQQSGAELVRPGASVTLSCKASG YIFTDYEMHWVKQTPVHGLEWIGAIDPETGGTAYNQKFKGKARLT ADKSSSTAYMELRSLTSEDSAVYYCTIYYTNHFDYWGQGTTLTVS SAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSG SLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPAS STTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPP NIKDVLMISLTPKVTCVVVDVSEDDPDVRISWFVNNVEVHTAQTQ THREDYNSTIRVVSALPIQHQDWMSGKEFKCKVNNKDLPSPIERT ISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVE WTSNGHTEENYKDTAPVLDSDGSYFIYSKLDIKTSKWEKTDSFSC NVRHEGLKNYYLKKTISRSPGK

[0067] Light chain (SEQ ID NO. 24 (removing signal peptide)):

TABLE-US-00008 MKLPVRLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASISCRSS QSLVYSNGDTYLYWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSG SGTDFTLKISRVEAEDLGVYFCSQSTHVPLTFGAGTKLELKRADA APTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQ NGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTS TSPIVKSFNRNEC

(v) Antibody 25F3C4

[0068] Heavy chain (SEQ ID NO. 25 (removing signal peptide)):

TABLE-US-00009 MEWSWVFLFLLAVIAGVQSQVQLQQSGAELVRPGASVTLSCKASG YTFTDYEVHWVKQTPVHGLEWIGAIDPETGGTAYNLKFKGKAILT ADKSSSTAYMELRSLTSEDSAVYYFTIYYTNNFDYWGQGTALTVS SAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSG SLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPAS STTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPP NIKDVLMISLTPKVTCVVVDVSEDDPDVRISWFVNNVEVHTAQTQ THREDYNSTIRVVSALPIQHQDWMSGKEFKCKVNNKDLPSPIERT ISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVE WTSNGHTEENYKDTAPVLDSDGSYFIYSKLDIKTSKWEKTDSFSC NVRHEGLKNYYLKKTISRSPGK

[0069] Light chain (SEQ ID NO. 26 (removing signal peptide)):

TABLE-US-00010 MKLPVRLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASISCRSS QSLVYSDGNTYLHWYLQKPGQSPKLLIYKVSYRFSGVPDRFSGSG SGTDFTLKISRVEAEDLGVYFCSQSTHVPLTFGAGTKLELKRAD AAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSER QNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKT STSPIVKSFNRNEC

[0070] In the sequences above, the part in bold refers to a signal peptide; the part in italic type refers to a variable region, wherein the underlined part denotes a CDR; and the underlined part behind the variable region is a constant region.

Embodiment 2 Screening and Verification of Capture and Detection Antibodies

(I) First Pairing and Screening

[0071] For the 5 monoclonal antibodies obtained in Embodiment 1, one antibody is paired with another as capture and detection antibodies, respectively, and the detection effect on the recombinant human REG1A of antigens is measured. The process is as follows: [0072] Coating: A diluent (10 mM pH 7.4 PBS) is used to dilute the capture antibody to 2.5 ?g/mL as a coating buffer, and the coating buffer is then added to the wells on an ELISA plate (100 ?L per well) to be incubated overnight at 4? C. After that, the liquid is removed from wells, and the plate is washed twice with a wash buffer (10 mM pH7.4 PBS+0.5% Tween 20, PBST); [0073] Blocking: A blocking buffer (10 mM pH7.4 PBS+1% BSA+0.5% Tween 20) is added to the wells on an ELISA plate (250 ?L per well) to be incubated for 2 h at 37? C. Then, the liquid is removed from wells, and the plate is washed twice with PBST; [0074] Antigen incubation: A basic diluent (10 mM pH7.4 PBS+0.5% BSA+0.5% Tween 20) is used to prepare the recombinant REG1A into solutions of different concentrations, and then the solutions are added to the wells of an ELISA plate (100 ?L per well) to be incubated for 1 h at 37? C. After that, the liquid is removed from wells, and the plate is washed 3 times with PBST; [0075] Detection antibody incubation: A basic diluent is used to dilute the biotin-labeled detection antibody to 1 ?g/mL, and then the solution is added to the wells of an ELISA plate (100 ?L per well) to be incubated for 1 h at 37? ? C. After that, the liquid is removed from wells, and the plate is washed 3 times with PBST; [0076] Secondary antibody incubation: A basic diluent is used to dilute the HRP-labeled streptavidin (SIGMA) by a factor of 20,000, and then the solution is added to the wells of an ELISA plate (100 ?L per well) to be incubated for 45 min at 37? C. After that, the liquid is removed from wells, and the plate is washed 3 times with PBST; [0077] Color developing: A TMB substrate solution (produced by Huzhou InnoReagents Co., Ltd.) is added to the wells of an ELISA plate (100 ?L per well) for color development for 15 min at 37? C. away from light. Then, a 50 ?L stop buffer (0.2M sulfuric acid) is added to stop the reaction; [0078] Reading: A microplate reader is used to measure the OD value of each well at the dominant wavelength of 450 nm and the sub-wavelength of 620/630 nm (the OD value at the dominant wavelength of 450 nm minus the OD value at the sub-wavelength of 620/630 nm).

[0079] The results are shown in Table 1.

TABLE-US-00011 TABLE 1 Results of the First Pairing Experiment (OD Value) Capture Detection antibody Antigen (ng/mL) antibody (2.5 ?g/mL) 100.00 33.33 11.11 3.70 1.23 0 (1 ?g/mL) 4H8F1 0.09 0.081 0.081 0.079 0.074 0.074 4H8F1-biotin 4.242 4.223 4.104 3.736 2.465 0.21 11D11B11-biotin 4.33 4.33 4.217 3.841 2.987 0.088 12F12A10-biotin 0.08 0.073 0.07 0.068 0.072 0.069 21B11D2-biotin 0.101 0.097 0.095 0.09 0.09 0.093 25F3C4-biotin 11D11B11 4.235 4.478 3.872 2.865 1.373 1.208 4H8F1-biotin 0.064 0.064 0.072 0.066 0.066 0.079 11D11B11-biotin 4.216 4.083 3.896 2.811 1.047 0.083 12F12A10-biotin 0.065 0.063 0.066 0.06 0.07 0.068 21B11D2-biotin 0.083 0.083 0.076 0.077 0.073 0.077 25F3C4-biotin 12F12A10 3.183 3.267 3.05 2.425 1.297 0.068 4H8F1-biotin 3.153 3.028 2.688 1.543 0.934 0.075 11D11B11-biotin 0.096 0.098 0.088 0.088 0.098 0.089 12F12A10-biotin 3.352 3.21 2.892 2.384 1.177 0.068 21B11D2-biotin 3.77 3.759 3.506 2.998 1.608 0.085 25F3C4-biotin 21B11D2 0.084 0.078 0.073 0.077 0.078 0.075 4H8F1-biotin 0.075 0.077 0.077 0.069 0.089 0.074 11D11B11-biotin 4.648 4.587 4.228 4.589 3.898 0.09 12F12A10-biotin 0.068 0.071 0.069 0.069 0.066 0.073 21B11D2-biotin 0.106 0.094 0.111 0.089 0.087 0.09 25F3C4-biotin 25F3C4 0.086 0.085 0.078 0.082 0.087 0.076 4H8F1-biotin 0.089 0.083 0.079 0.076 0.071 0.074 11D11B11-biotin 4.476 4.289 4.373 4.251 3.764 0.093 12F12A10-biotin 0.073 0.071 0.069 0.068 0.066 0.068 21B11D2-biotin 0.107 0.118 0.103 0.102 0.099 0.093 25F3C4-biotin

[0080] According to Table 1, compared with other sets of paired antibodies, the following three sets of paired antibodies can obtain a better detection effect: separately used as 4H8F1, 21B11D2 and 25F3C4 of the capture antibody and as 12F12A10 of the detection antibody.

(II) Second Pairing and Screening

[0081] The three sets of paired antibodies obtained from the first pairing and screening are further screened. The process is as follows:

[0082] Coating: A PBS is used to dilute the capture antibody to 1 ?g/mL as a coating buffer, and then the coating buffer is added to the wells on an ELISA plate (100 ?L per well) to be incubated overnight at 4?C. After that, the liquid is removed from wells, and the plate is washed twice with PBST;

[0083] Blocking: A blocking buffer is added to the wells on an ELISA plate (250 ?L per well) to be incubated for 2 h at 37? C. After that, the liquid is removed from wells, and the plate is washed twice with PBST;

[0084] Antigen incubation: A basic diluent is used to prepare the recombinant REG1A into solutions of different concentrations, and then the solutions are added to the wells of an ELISA plate (100 ?L per well) to be incubated for 1 h at 37? C. After that, the liquid is removed from wells, and the plate is washed 3 times with PBST;

[0085] Detection antibody incubation: A basic diluent is used to dilute the HRP-labeled detection antibody by a factor of 30,000, and then the solution is added to the wells of an ELISA plate (100 ?L per well) to be incubated for 1 h at 37? C. After that, the liquid is removed from wells, and the plate is washed 3 times with PBST;

[0086] Color developing: A TMB substrate solution is added to the wells of an ELISA plate (100 ?L per well) for color development for 15 min at 37? C. away from light. Then, a 50 ?L stop buffer (0.2M sulfuric acid) is added to stop the reaction;

[0087] Reading: A microplate reader is used to measure the OD value of each well at the dominant wavelength of 450 nm and the sub-wavelength of 620/630 nm (the OD value at the dominant wavelength of 450 nm minus the OD value at the sub-wavelength of 620/630 nm).

[0088] The results are shown in Table 2 and FIG. 2.

TABLE-US-00012 TABLE 2 Results of the Second Pairing Experiment (OD Value) Detection antibody 12F12A10 Coated antibody 4H8F1 21B11D2 25F3C4 REG1A 1000 1.3618 3.0054 2.9005 pg/mL 500 0.9267 2.8918 2.8358 250 0.5655 2.3096 2.1300 125 0.3233 1.3078 1.2050 62.5 0.2205 0.5776 0.5386 31.25 0.1925 0.2769 0.2784 15.625 0.2028 0.1943 0.2411 0 0.2394 0.1382 0.1769

[0089] It can be seen from the results that the antibody pairs21B11D2-12F12A10 and 25F3C4-12F12A10show a good linearity within the range that the concentration of REG1A is less than 500 pg/mL, and the antibody pair4H8F1-12F12A10shows the optimal linearity when the concentration exceeds 1,000 pg/mL. Considering that the concentration of REG1A in human serum samples is Level ng, the antibody pair4H8F1-12F12A10is finally used as the capture and detection antibodies.

(III) Verification of Capture and Detection Antibodies

[0090] 4H8F1, 21B11D2 and 25F3C4 are used separately used as coated antibodies and 12F12A10 is used as the detection antibody to test serum samples. The steps in (II) Second pairing and screening above are followed. When serum samples are tested, antigens are replaced with serum samples that are then added to the wells (100 ?L per well) directly. The concentration of proteins in serum samples relative to the standard curve are calculated.

[0091] The results are shown in Table 3 and FIG. 3.

TABLE-US-00013 TABLE 3 Results of Serum Detection of REG1A Antibody Pairs Detection antibody Coated antibody Standard protein 12F12A10 concentration 4H8F1 21B11D2 25F3C4 (ng/ml) Value Standard 100 2.8884 2.2908 2.6976 curve 50 2.6029 2.2536 2.6941 10 2.2273 2.1506 2.6155 1 1.4997 1.1509 1.6932 0.1 0.6664 0.1889 0.1995 0 0.0589 0.1013 0.0693 No. Concentration (ng/ml) Serum 170 84.0 4.1 2.3 samples 232 81.2 2.6 3.4 242 43.9 2.9 6.3 253 2.8 24.9 1.1

[0092] It can be seen from the standard curve that the two antibody pairs21B11D2-12F12A10 and 25F3C4-12F12A10show a small change in the OD values when the antigen concentration is equal to or greater than 10 ng/ml, speculating that the antigen-antibody response in this case has reached the saturation point. This point can also be demonstrated by the detection results of serum samples, and for the two antibody pairs, the measured concentrations of proteins in serum samples are not higher than 10 ng/ml. Taking samples 232 and 242 as an example, the measured concentrations of proteins of these two samples4H8F1-12F12A10vary greatly, but the concentrations detected of the two antibody pairs21B11D2-12F12A10 and 25F3C4-12F12A10show no significant differences.

Embodiment 3 Adoption of the Antibody Pair4H8F1-12F12A10for Measurement of REG1A in Different Samples

[0093] The serum samples are taken from 805 normal cases and the serum samples from 1,924 patients with colorectal cancer (CRC) (including 344 patients with Stage 1 CRC, 669 patients with Stage 2 CRC, 567 patients with Stage 3 CRC and 344 patients with Stage 4 CRC), 89 patients with gastric cancer, 198 patients with adenomatous polyp, 34 patients with IBD, 80 patients with peptic ulcer, 158 patients with gastroenteritis and 7 patients with acute pancreatitis. These serum samples are obtained from Sun Yat-sen University Cancer Center (SYSUCC) and Zhujiang Hospital of Southern Medical University.

[0094] As described above, the antibody combination (4H8F1-12F12A10) provided by the present invention is used to test serum samples, and 4H8F1 and 12F12A10 are used as a coated antibody (capture antibody) and a detection antibody respectively to prepare an ELISA kit. Normal cases are used as a control group whose data are compared with the data of the disease group, to conduct a ROC analysis. Then, the results of ROC analysis and the needs of clinical application are combined to determine the cutoff value of concentration of REG1A in human serum as 60 ng/ml, and based on it, the corresponding sensitivity and specificity are calculated. The results are shown in Table 4.

TABLE-US-00014 TABLE 4 Detection of the Specificity and Sensitivity of Different Serum Samples (Unit of Protein Concentration: ng/ml) Mean protein Total number Cutoff: 60 concentration T-test AUC P value of cases Negative Positive Sensitivity Specificity Normal cases 46.42 805 671 134 83.35% CRC 1 90.87 2.72E?28 0.9038 <0.0001 344 86 258 75.00% CRC 2 94.42 1.68E?41 0.9007 <0.0001 669 158 511 76.38% CRC 3 96.97 1.92E?42 0.9147 <0.0001 567 116 451 79.54% CRC 4 112.08 2.08E?19 0.8997 <0.0001 344 75 269 78.20% CRC ALL 96.36 1.04E?110 0.8946 <0.0001 1924 477 1447 75.21% Gastric cancer 146.42 6.25E?09 0.9033 <0.0001 89 19 70 78.65% Adenomatous polyp 76.85 1.27E?15 0.7672 <0.0001 198 77 121 61.11% IBD 103.16 1.41E?07 0.9059 <0.0001 34 8 26 76.47% Peptic ulcer 118.33 6.55E?10 0.8518 <0.0001 80 25 55 68.75% Gastroenteritis 102.38 3.28E?24 0.8705 <0.0001 158 39 119 75.32% Acute pancreatitis 334.58 3.87E?02 0.9807 <0.0001 7 0 7 100.00% Total sensitivity 75.53%

[0095] According to the measured data above, GraphPad Prism is used to draw a receiver operating characteristic (ROC) curve, and the results are shown in FIG. 4.

Embodiment 4 Adoption of the Antibody Pair4H8F1-12F12A10for Measurement of REG1A in Samples Before and After the Treatment of Patients with Inflammatory Bowel Disease (IBD)

[0096] The serum samples of 29 patients with IBD before and after the effective treatment of IBD are obtained from SYSUCC. Infliximab and other monoclonal antibodies or hormones that are often used clinically as a treatment for IBD are used to treat the patients with IBD. The confirmation of treatment efficacy is made by the attending physician according to the endoscopic score. The prevalence situation of patients with IBD is confirmed though enteroscopy, and in the absence of any therapeutic intervention, the serum samples before treatment are obtained. After patients receive medication for 3 months and the treatment is confirmed to be effective, the serum samples after treatment are obtained.

[0097] As described above, the antibody combination (4H8F1-12F12A10) provided by the present invention is used to test serum samples, and 4H8F1 and 12F12A10 are used as a coated antibody (capture antibody) and a detection antibody respectively to prepare an ELISA kit, and the concentrations of REG1A in serum samples before and after the effective treatment are tested.

[0098] Through the test of the serum samples taken from 29 patients with active IBD, it is found that the concentration of REG1A in serum of 26 patients (89.7%; P<0.0001) after effective treatment is significantly reduced, and the median concentration in serum decreases from 85.86 ng/ml (31.68-317.80) before effective treatment to 60.20 ng/ml (29.34-171.50). For the results, see FIG. 5.

[0099] Experiments prove that the antibody pair4H8F1-12F12A10provided by the present invention can be used to conduct the companion diagnostics/prognostic monitoring of IBD and help confirm whether the therapeutic drugs for patients with IBD are effective to avoid repeated colonoscopies.

[0100] The foregoing description of the preferred embodiments of the present invention is not limited to the present invention. Those skilled in the field may make various changes or deformations according to the present invention, and all changes or deformations are therefore intended to be embraced in the Claims of the present invention, without departing from the spirit thereof.