Anti-CALR mutant protein antibody, preparation method and detection kit

Abstract

The present invention relates to the biological field and discloses an anti-CALR mutant protein antibody, which comprises a framework region and a complementary determining region; the complementary determining region comprises CDR-VH1, CDR-VH2 and CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL3; the complementary determining region CDR-VH1 sequence is SEQ ID NO.1; the complementary determining region CDR-VH2 sequence is SEQ ID NO.2; the complementary determining region CDR-VH3 sequence is SEQ ID NO.3; the complementary determining region CDR-VL1 sequence is SEQ ID NO.4; the complementary determining region CDR-VL2 sequence is SEQ ID NO.5; the complementary determining region CDR-VL3 sequence is SEQ ID NO.6.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody preparation and immunological detection, and in particular to an anti-CALR (myeloproliferative neoplasm marker calreticulin) mutant protein antibody, a preparation method and a detection kit Background Art

[0002] Myeloproliferative neoplasms (MPNs) are a type of clonal, chronic proliferative diseases originating from bone marrow hematopoietic stem cells, mainly including polycythemia vera (PV), thrombocythemia (ET) and myelofibrosis (MF).

[0003] In recent years, mutations in multiple molecular markers such as JAK2, MPL, and TET have been discovered, which are of great significance for the detection and diagnosis of MPN. However, studies have shown that 30%-45% of ET or MF patients with wild-type JAK2 / MPL still have difficulty in diagnosis.

[0004] In recent years, many studies have shown that characteristic calreticulin (CALR) mutations were found in MPN patients with negative JAK2 mutations, suggesting that calreticulin (CALR) mutations are expected to become another new molecular marker for the diagnosis of myeloproliferative neoplasms.

[0005] Current studies have shown that there are more than 50 types of CALR mutations, the most common of which are the deletion of 52 bases in exon 9 (p.L367fs*46, type I mutation) and the insertion of 5 bases TTGTC (p.K385fs*47, type II mutation). The number of patients carrying these two types of mutations accounts for about 80% of the number of patients carrying all CALR mutations. Although there are many types of deletion or insertion mutations in CALR exon 9, no matter what form of mutation, it will lead to a base pair reading frame shift, and then produce a new C-carboxyl terminal protein lacking the endoplasmic reticulum retention sequence (KDEL amino acid sequence). The detection of this CALR mutant peptide may become a new idea for clinical detection of CALR mutations.

[0006] At present, studies have prepared polyclonal antibodies and monoclonal antibodies (CAL2) based on this characteristic of CALR mutant protein for immunohistochemical staining of bone marrow tissue of MPN patients. Polyclonal antibodies have nonspecific staining, and although CAL2 is a monoclonal antibody, it can only be used for immunohistochemical detection of formalin-fixed bone marrow wax blocks. The analysis of the results is heavily dependent on the subjective judgment of technicians, and standardized detection of markers and disease diagnosis cannot be achieved. Summary of the invention

[0007] The technical problem to be solved by the present invention is that, in view of the above-mentioned defects of the prior art, an anti-CALR mutant protein antibody is provided, wherein the antibody comprises a framework region and a complementary determining region; the complementary determining region comprises CDR-VH1, CDR-VH2 and CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL3; the complementary determining region CDR-VH1 sequence is SEQ ID NO.1; the complementary determining region CDR-VH2 sequence is SEQ ID NO.2; the complementary determining region CDR-VH3 sequence is SEQ ID NO.3; the complementary determining region CDR-VL1 sequence is SEQ ID NO.4; the complementary determining region CDR-VL2 sequence is SEQ ID NO.5; the complementary determining region CDR-VL3 sequence is SEQ ID NO.6.

[0008] As an improvement to the anti-CALR mutant protein antibody, the amino acid sequence of the heavy chain complementary determining region of the antibody is SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, or an amino acid sequence with equivalent function formed by replacing, deleting or adding one or more amino acids in the sequence; the amino acid sequence of the light chain complementary determining region is SEQ ID NO.4, SEQID NO.5, SEQ ID NO.6, or an amino acid sequence with equivalent function formed by replacing, deleting or adding one or more amino acids in the sequence.

[0009] As a further improvement of the anti-CALR mutant protein antibody, the antibody includes heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H whose sequences are shown in SEQ ID NOs: 7-10; and light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L whose sequences are shown in SEQ ID NOs: 11-14.

[0010] As a further improvement of the anti-CALR mutant protein antibody, the antibody further comprises a constant region.

[0011] As a further improvement to the anti-CALR mutant protein antibody, the constant region is selected from any one of the constant regions of IgG1, IgG2, IgG3, and IgG4.

[0012] As a further improvement to the anti-CALR mutant protein antibody, the species origin of the constant region is cattle, sheep, rabbit, mouse, rat or human.

[0013] A method for preparing an anti-CALR mutant protein antibody comprises the following steps: S1, designing and synthesizing a dominant antigen epitope polypeptide based on the abnormal amino acid segment at the C-carboxyl terminal of the CALR mutant protein, coupling the polypeptide to a carrier protein to form a complete antigen; S2, mixing the complete antigen prepared in the previous step with Freund's complete adjuvant in equal volumes and emulsifying them evenly; after multiple immunizations of Balb / c mice, collecting blood from the eye sockets of each mouse, and separating serum by centrifugation; S3, determining the serum titer, and selecting the serum with a titer greater than 10 7 The mice were given intrasplenic booster immunization; S4 after the booster immunization, the spleen cells were taken and fused with sp2 / 0 myeloma cells in proportion; S5 after the fusion, the culture medium was added to terminate the fusion, and the cells were resuspended in the culture medium and cultured in an incubator; S6 after the fusion cell culture, the specific antibodies in the cell culture supernatant were detected to obtain hybridoma cells; S7 healthy mice were taken, paraffin was injected intraperitoneally and then hybridoma cells were injected respectively, and the ascites was collected to obtain monoclonal antibodies.

[0014] Use of an anti-CALR mutant protein antibody in preparing a reagent for detecting thrombocythaemia and / or myelofibrosis.

[0015] A kit for detecting thrombocythemia and myelofibrosis, comprising a coated antibody and a labeled antibody; the coated antibody is the monoclonal antibody as claimed in claim 1, 2 or 3; the labeled antibody is a monoclonal antibody targeting the N-terminus of CALR protein.

[0016] As an improvement to the thrombocythemia and myelofibrosis detection kit, the kit is any one of an immunohistochemistry kit, an enzyme-linked immunosorbent assay kit, a chemiluminescence immunoassay kit, a colloidal gold immunoassay kit, an immunofluorescence kit, and a flow cytometry kit.

[0017] According to the above technical solution, the present invention has the following beneficial effects:

[0018] The novel monoclonal antibody against CALR mutant protein provided in the present application has a completely new antigen binding domain, specifically binds only to CALR mutant protein and does not bind to wild-type CALR protein, and has the advantages of strong activity and affinity, high stability, etc.

[0019] The above monoclonal antibody and kit have strong specificity and high sensitivity when used for the detection of serum CALR mutant protein, and the detection range reaches 2.93-375ng / mL.

[0020] The above-mentioned detection reagents can effectively promote the application of CALR mutant protein detection and promote the inclusion of CALR mutant protein detection in routine clinical testing, and play an important role in the clinical diagnosis of thrombocythaemia and myelofibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following briefly introduces the drawings required for use in the embodiments:

[0022] Figure 1 Monoclonal antibody antigen recognition specificity detection (western blot);

[0023] Figure 2 Immunohistochemical staining with monoclonal antibodies against CALR mutant proteins;

[0024] Figure 3 CALR mutant enzyme-linked immunosorbent assay (ELISA) kit standard curve;

[0025] Figure 4 An example of detecting the concentration of CALR mutant protein in 20 clinical serum samples using an enzyme-linked immunosorbent assay (ELISA) kit;

[0026] Figure 5 Schematic diagram of the interpretation method of CALR mutant cell standards detected by flow cytometry kit. DETAILED DESCRIPTION

[0027] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings, in which typical embodiments of the present invention are shown.

[0028] Example 1 Preparation and identification of monoclonal antibodies against CALR mutant proteins

[0029] 1.1 Immunogen design and preparation

[0030] Aiming at the abnormal amino acid segment at the C-carboxyl terminus of the CALR mutant protein, a dominant antigen epitope peptide CALR-1 (SPARPRTSCREACLQGWTEA) was designed based on theoretical studies on the hydrophilicity, surface accessibility, and antigenic tendency of each amino acid segment, and a wild-type carboxyl terminal antigen epitope peptide CALR-2 (EDEEDEEDKEEDEEEDVPGQAKDEL) was designed as a control;

[0031] The peptides were coupled with keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) using the glutaraldehyde method to form complete antigens (CALR-1-KLH, CALR-2-KLH, CALR-1-BSA, CALR-2-BSA).

[0032] 1.2 Immunization of mice

[0033] The CALR-1-KLH complete antigen prepared in the previous step was mixed with Freund's complete adjuvant in equal volumes and emulsified evenly;

[0034] Six-week-old Balb / c mice were immunized by subcutaneous injection at multiple sites for three times, with an interval of 2 weeks between each immunization;

[0035] Two weeks after the third immunization, blood was collected from the eye sockets of each mouse, and the serum was separated by centrifugation. The serum titer was determined by indirect ELISA, and the serum titer greater than 10 was selected. 7 Mice were given an intrasplenic boost immunization.

[0036] 1.3 Cell fusion

[0037] Three days after booster immunization, spleen cells were fused with sp2 / 0 myeloma cells at a ratio of 6:1, using PEG (MW1450, Sigma) as the fusion agent;

[0038] After 10 minutes of fusion, fresh serum-free medium was added to terminate the fusion, and the cells were resuspended in DMEM culture medium containing 2% HAT and cultured in a 37°C, 5% CO2 incubator.

[0039] 1.4 Clone screening

[0040] The fused cells were cultured for 7-10 days, and the specific antibodies in the cell culture supernatant were detected by indirect method using 96-well plates coated with CALR-1-BSA and CALR-2-BSA;

[0041] If the supernatant of the same cell line is positive only when reacting with CALR-1-BSA, but negative when reacting with CALR-2-BSA, the cell line is determined to be a hybridoma cell line that can secrete antibodies that specifically recognize CALR mutant proteins;

[0042] After three consecutive subclonings, when the antibody positivity rate detected by ELISA was 100%, it was determined to be a hybridoma cell that stably expressed the target antibody. Finally, three hybridoma cells with strong specificity and high titer were obtained and named Cal26B10, Cal26B11, and Cal26B15, and preserved.

[0043] 1.5 Preparation and purification of mouse monoclonal antibodies

[0044] Healthy F1 mice were intraperitoneally injected with paraffin wax (0.5 ml / mouse). Seven days later, Cal26B10, Cal26B11, and Cal26B15 cells were injected at a dose of 10 7 After 7-10 days, the ascites was collected and purified by caprylic acid-ammonium sulfate and Protein A affinity purification to obtain a high purity (>95%) mouse monoclonal antibody.

[0045] 1.6 Specific identification of anti-CALR mutant protein monoclonal

[0046] Peripheral blood was collected from patients with thrombocythemia (ET) and myelofibrosis (MF). Peripheral blood mononuclear cells were separated by Ficoll and total protein was extracted. Western blot was used to detect the specificity of the antibody. Total protein of 293T cells stably transfected with CALR wild-type plasmid by lentivirus was used as negative control, and total protein of 293T cells stably transfected with CALR type I mutant and CALR type II mutant plasmid was used as positive control.

[0047] Western blot results showed (eg Figure 1 As shown in the figure, Cal26B10, Cal26B11, and Cal26B15 can react with clinical peripheral blood mononuclear cell samples positive for CALR mutation and 293T cell samples overexpressing mutant CALR protein, but do not react with 293T cell samples overexpressing wild-type CALR protein, indicating that the antibodies have good specificity.

[0048] Figure 1 In (results of antibody preparation specificity detection), WT: 293T cells overexpressing wild-type CALR; del52: 293T cells overexpressing CALR type I mutation; ins5: 293T cells overexpressing CALR type II mutation; P1: peripheral blood PBMC cells of MPN patients carrying CALR type I mutation; P2: peripheral blood PBMC cells of MPN patients carrying CALR type II mutation.

[0049] Example 2 Affinity determination of monoclonal antibodies against CALR mutant proteins

[0050] The affinity of each antibody was determined using WeSPR 100, and the results are shown in Table 1 (anti-CALR mutant protein monoclonal antibody affinity test results). Cal26B11 with the highest affinity was selected as the anti-CALR mutant protein monoclonal antibody for detection and used for subsequent testing and further research and development.

[0051] Ka(M-1s-1) Kd(s-1) KD(M) Cal26B10 <![CDATA[1.24×10 5 ]]> <![CDATA[6.80×10 -4 ]]> <![CDATA[5.48×10 -9 ]]> Cal26B11 <![CDATA[3.00×10 5 ]]> <![CDATA[1.77×10 -4 ]]> <![CDATA[5.90×10 -10 ]]> Cal26B15 <![CDATA[4.48×10 5 ]]> <![CDATA[3.36×10 -4 ]]> <![CDATA[7.51×10 -10 ]]>

[0052] Table 1 Affinity test results of monoclonal antibodies against CALR mutant proteins

[0053] Example 3 Analysis of the variable region of Cal26B11 monoclonal antibody

[0054] 3.1 Gene fishing

[0055] The monoclonal antibody Cal26B11 hybridoma cell line was expanded and 10 7cells, extracted total cellular RNA, obtained cDNA products by reverse transcription, amplified and recovered by rTaq DNA polymerase, inserted into pMD-18T vector, transformed into DH5α competent cells, and after colonies grew, 3 heavy chain and light chain gene clones were taken and sent to a gene sequencing company for sequencing.

[0056] 3.2 Sequence analysis of heavy and light chain variable region genes

[0057] The nucleotide sequence (345 bp) encoding the heavy chain variable region of the monoclonal antibody Cal26B1 is as follows:

[0058] CAGATCCAGTTGGTGCAGTCTGGACCTGTTCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAAACTATGGAATGACCTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAAAGC CAACATATGCTGGTGACTTCAAGGGACGATTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACGACCTCAAAAATGAGGACACGGCTACATATTTCTGTTCAAGCCAATTACGAGGGGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0059] Heavy chain variable region CDRH1 amino acid sequence:

[0060] GYTFTNY (SEQ ID NO: 1)

[0061] Heavy chain variable region CDRH2 amino acid sequence:

[0062] NTYTGK (SEQ ID NO: 2)

[0063] Heavy chain variable region CDRH3 amino acid sequence:

[0064] FCSSQLRGAY (SEQ ID NO: 3)

[0065] Heavy chain framework region amino acid sequence:

[0066] FR1-H:QIQLVQSGPVLKKPGETVKISCKAS(SEQ ID NO:7)

[0067] FR2-H:GMTWVKQAPGKGLKWMGWI(SEQ ID NO:8)

[0068] FR3-H:PTYAGDFKGRFAFSLETSASTAYLQINDLKNEDTATY(SEQ ID NO:9)

[0069] FR4-H:WGQGTLVTVSA(SEQ ID NO:10)

[0070] The nucleotide sequence (339 bp) encoding the light chain variable region of monoclonal antibody C8F3 is as follows:

[0071] GACATTGTGATGTCACAGTCTCCGTCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTACCAATCAGAAGAACTCCTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGG CATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTTATTACTGTCAGCAATATTATAGCTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

[0072] Light chain variable region CDRL1 amino acid sequence:

[0073] KSSQSLLYSTNQKNSLA(SEQ ID NO:4)

[0074] Light chain variable region CDRL2 amino acid sequence:

[0075] WASTRES (SEQ ID NO: 5)

[0076] Light chain variable region CDRL3 amino acid sequence:

[0077] CQQYYSYPWT (SEQ ID NO: 6)

[0078] Light chain framework region amino acid sequence:

[0079] FR1-L:DIVMSQSPSSLAVSVGEKVTMSC(SEQ ID NO:11)

[0080] FR2-L:WYQQKPGQSPKLLIY(SEQ ID NO:12)

[0081] FR3-L:GVPDRFTGSGSGTDFTLTISSVKAEDLAVYY(SEQ ID NO:13)

[0082] FR4-L: FGGGTKLEIK(SEQ ID NO:14)

[0083] Example 4 Immunohistochemical staining study of Cal26B11 monoclonal antibody and human MPN samples

[0084] 4.1 Clinical sample collection

[0085] Bone marrow tissue blocks of 28 patients with MPN (PV, ET, MF) were collected from the outpatient clinics and inpatient wards of Huashan Hospital North Branch and Changhai Hospital from 2015 to 2017;

[0086] Among them, 5 cases carried CALRI mutation, 4 cases carried CALRII mutation, 1 case carried CALR V (c.1091_1142del) mutation, and 18 cases carried JAK2 mutation. The collected clinical samples were immunohistochemically stained with Cal26B11 antibody;

[0087] The results of immunohistochemical staining were scored according to the ASCO / CAP guidelines (American Society of Clinical Oncology / College of American Pathologists).

[0088] 4.2 Immunohistochemical staining

[0089] (1) Select tissue wax blocks from MPN patients and obtain 4 μm tissue paraffin sections.

[0090] (2) Dewax with xylene twice at room temperature, 10 minutes each time.

[0091] (3) Wash in ethanol gradient to remove xylene, and then wash in double distilled water for 5 minutes.

[0092] (4) Use 0.3% H 2 O 2 Block endogenous peroxidase activity for 10 minutes at room temperature.

[0093] (5) Wash in double distilled water 4 times, 5 minutes each time.

[0094] (6) Cal26B11 monoclonal antibody was added dropwise to the tissue sections and incubated at 4°C for 12 hours.

[0095] (7) Wash with PBS buffer 4 times, 5 minutes each time.

[0096] (8) Add horseradish peroxidase-labeled secondary antibody and incubate at 37°C for 30 min.

[0097] (9) Wash with PBS buffer 4 times, 5 minutes each time.

[0098] (10) Add DAB colorimetric reagent and incubate at room temperature for color development.

[0099] (11) Wash with double distilled water 4 times, 5 minutes each time.

[0100] (12) Allow to dry, seal the slides, and observe under a microscope.

[0101] The results showed that the immunohistochemical staining results were completely consistent with the Sanger sequencing results, and only samples with CALR mutations were positively stained, while samples without CALR mutations were not stained ( Figure 2 ).

[0102] Sample No. Clinical diagnosis Sanger sequencing CALR mutant immunohistochemistry results P1 PV JAK2 V617F — P2 PV JAK2 V617F — P3 PV JAK2 V617F — P4 PV JAK2 V617F — P5 PV JAK2 V617F — P6 PV JAK2 V617F — P7 PV JAK2 V617F — P8 ET CALR Type I Megakaryocytes (3+) P9 ET CALR Type I Polymorphonuclear granulocytes (1+) P10 ET JAK2 V617F — P11 ET CALR type II Megakaryocytes (1+) P12 ET JAK2 V617F — P13 ET JAK2 V617F — P14 ET CALR Type I Megakaryocytes (2+) Polymorphonuclear granulocytes (1+) P15 ET JAK2 V617F — P16 ET CALR Type I Polymorphonuclear granulocytes (1+) P17 ET JAK2 V617F — P18 ET JAK2 V617F — P19 ET CALR V-Type Megakaryocytes (3+) Polymorphonuclear granulocytes (1+) P20 ET CALR type II Polymorphonuclear granulocytes (1+) P21 ET JAK2 V617F — P22 ET CALR type II Polymorphonuclear granulocytes (1+) P23 PMF JAK2 V617F — P24 PMF CALR Type I PMN(1+) P25 PMF JAK2 V617F — P26 PMF JAK2 V617F — P27 PMF CALR type II PMN(1+) P28 PMF JAK2 V617F —

[0103] Table 2 Clinical diagnosis, genetic testing and pathological analysis results of 28 MPN patients

[0104] Figure 2 , Cal26B11 antibody was used for immunohistochemical staining of bone marrow sections of MPN patients (×200 times) AI corresponds to the relevant sample number in Table 2:

[0105] A: The corresponding sample number is P8;

[0106] B: The corresponding sample number is P9;

[0107] C: The corresponding sample number is P11;

[0108] D: The corresponding sample number is P14;

[0109] E: The corresponding sample number is P19;

[0110] F: The corresponding sample number is P20;

[0111] G: The corresponding sample number is P22;

[0112] H: The corresponding sample number is P24;

[0113] I: The corresponding sample number is P28.

[0114] Example 5 Establishment of CALR mutant protein detection kit

[0115] The kit contains the coating antibody Cal26B11 and the labeling antibody C7492 (Sigma-Aldrich). The kit is an enzyme-linked reaction kit. The specific operation steps are as follows:

[0116] The coating antibody Cal26B11 was diluted to 10 μg / ml with CB, and 100 μl was added to each well of the ELISA plate and coated overnight at 4°C;

[0117] The next day, the ELISA plate was patted dry, and 200 μl of blocking solution (PBS containing 1% BSA) was added to each well. The plate was blocked at 37°C for 2 h, and then patted dry after the blocking was completed.

[0118] Add 100 μl of standard or patient serum sample (patient serum sample needs to be diluted 3 times) to each well;

[0119] After incubation at 37°C for 1 h, the plate was washed three times with 0.05% PBST;

[0120] Dilute the enzyme-labeled antibody to a 1000-fold working solution with a washing solution containing 1% BSA, add 50 μl to each well, incubate at 37°C for 30 min, then wash the plate 5 times and pat dry;

[0121] Add 100 μl of TMB colorimetric solution to each well and incubate at 37°C for 10 min;

[0122] Add 50 μl of stop solution to each well and read the OD value at 450 nm on a microplate reader.

[0123] 5.1 Kit detection linearity

[0124] The CALR mutation standard protein (375 ng / mL) was diluted 2-fold with negative serum to 2.93 ng / mL, and each gradient dilution sample was tested, the absorbance value was measured and a standard curve was drawn. The P / N value greater than or equal to 2 was regarded as a positive result, and the lowest value higher than twice the blank OD value was regarded as the detection limit. The results are shown in Table 3 and Figure 3 shown.

[0125]

[0126]

[0127] Table 3. Linearity detection of paired antibodies

[0128] According to the above Table 3 and Figure 3The experimental data show that the kit obtained by using the coating antibody Cal26B11 and the labeling antibody C7492 (Sigma-Aldrich) in this application still has obvious positive performance at an antigen concentration as low as 2.93ng / mL, and can show a good linear relationship between 2.93-375ng / mL, and the fitting coefficient is greater than 0.99. This proves that the monoclonal antibody used in this application has a low detection limit, high sensitivity in practical application, and has broad application prospects in qualitative detection.

[0129] 5.2 Kit Detection Precision

[0130] The CALR mutation standard protein was added to the negative serum at a certain concentration, and the reagent intra-batch difference analysis was performed. Each sample was tested in parallel 3 times, and the intra-batch CV was less than 5%, and the intra-batch precision met the requirements.

[0131] The CALR mutation standard protein was added to the negative serum at a certain concentration, and the test was carried out with 3 batches of reagents for 5 consecutive days. The batch difference analysis of the reagents was performed, and the batch CV was less than 3%, and the batch precision met the requirements (Table 4).

[0132]

[0133] Table 4 Standard concentrations and corresponding absorbance (OD) values

[0134] 5.3 Kit Detection Specificity

[0135] The kit was used to simultaneously detect CALR mutant protein standards and CALR wild-type protein standards (187.5 ng / mL, 46.88 ng / mL, and 11.72 ng / mL) at the same concentrations. The t-test was used for statistical analysis, and p < 0.05. The results showed that the established CALR mutant protein ELISA kit had good specificity (Table 5).

[0136]

[0137] Table 5 Standard concentrations and corresponding absorbance (OD) values

[0138] 5.4 Testing of clinical samples with the kit

[0139] Ten sera from patients with CALR mutations (type I: J1522, J1525, J1526, J1531, J1535) and type II: J1550, J1567, J1576, J1580, J1675), five sera from patients with JAK2 mutations (J1504, J1505, J1516, J1538, J1618), and five serum samples from negative patients (P1-P5) were selected and tested using the CALR mutant protein ELISA reagent established above. The measured CALR concentrations were significantly different from those of the JAK2 group or the wild-type group (Table 6, Figure 4 ).

[0140] Figure 4 In the study, ELISA kits were used to detect the sera of 10 CALR mutation MPNs, 5 JAK2 mutation MPNs and 5 normal physical examination volunteers. The concentration of CALR mutant protein in peripheral serum of patients with CALR mutation confirmed by molecular detection methods was significantly higher than that of JAK2 mutation MPNs and normal physical examination volunteers.

[0141] It can be seen from the above experimental data that the CALR mutant protein ELISA reagent in the present application has good sensitivity, specificity, and high intra-batch and inter-batch precision, which proves that the above kit has excellent performance.

[0142] Patient number Absorbance value CALR protein concentration Patient number Absorbance value CALR protein concentration J1522 0.1371 20.84 J1504 0.0883 4.56 J1525 0.1321 18.51 J1505 0.0832 4.11 J1526 0.1082 7.4 J1516 0.0813 3.94 J1531 0.1221 13.86 J1538 0.0749 3.37 J1535 0.1652 33.91 J1618 0.0781 3.65 J1550 0.1711 36.65 P1 0.0577 1.84 J1567 0.1457 24.84 P2 0.08 3.82 J1576 0.1346 19.67 P3 0.0908 4.78 J1580 0.2216 60.14 P4 0.0697 2.91 J1675 0.1639 33.3 P5 0.0675 2.71

[0143] Table 6 Absorbance values ​​and corresponding CALR protein concentrations of clinical samples

[0144] Example 6 Establishment of a flow cytometry detection kit for CALR mutant protein

[0145] The kit contains the CALR mutant protein-specific binding antibody Cal26B11 (primary antibody) and the fluorescently labeled secondary antibody goat anti-mouse IgG Alexa Fluor 488 (abcam). The kit is a flow cytometry detection kit. The specific operation steps are as follows:

[0146] The cells were collected by trypsin digestion and centrifuged at 300 g for 6 min;

[0147] Remove the supernatant, add 1 ml PBS to wash, centrifuge at 300 g for 6 minutes, and remove the supernatant;

[0148] Every 10 6 The cells were fixed by adding 100 μl of 4% paraformaldehyde and mixed;

[0149] Incubate at room temperature for 20 minutes;

[0150] Add 1 ml PBS to wash the cells, centrifuge at 300 g for 6 minutes, remove the supernatant, and repeat once (wash the cells twice, i.e., repeat once by adding 1 ml PBS to wash the cells, centrifuge at 300 g for 6 minutes, and remove the supernatant);

[0151] Every 10 6 The cells were permeated by adding 100 μl of pre-cooled 90% methanol and incubated on ice for 15 min;

[0152] Add 1 ml PBS to wash, centrifuge at 300 g for 6 minutes, remove the supernatant, and repeat once (add 1 ml PBS to wash cells, centrifuge at 300 g for 6 minutes, remove the supernatant);

[0153] Add 100 μl of primary antibody at a concentration of 10 μg / ml, mix thoroughly, and incubate at 4°C in the dark for 1 hour;

[0154] Add 1 ml PBS to wash, centrifuge at 300 g for 6 minutes, remove the supernatant, and repeat once (wash the cells twice, i.e., repeat once adding 1 ml PBS to wash the cells, centrifuge at 300 g for 6 minutes, and remove the supernatant);

[0155] Add secondary antibody at a concentration of 0.5 μg / ml, mix thoroughly, and incubate at 4°C in the dark for 30 minutes;

[0156] Add 1 ml PBS to wash, centrifuge at 300 g for 6 minutes, remove the supernatant, and repeat once (wash the cells twice, i.e., repeat once by adding 1 ml PBS to wash the cells, centrifuge at 300 g for 6 minutes, and remove the supernatant).

[0157] Add 350 μl of PBS to resuspend the cells and detect them on a flow cytometer. The results are shown in Figure 5 , Figure 5 HEK-293T cells were used to overexpress mCherry empty vector, mCherry-CALR wild-type fusion protein, mCherry-CALR T1 mutant fusion protein, and mCherry-CALR T2 mutant fusion protein to prepare cell standards. Flow cytometry was used to detect CALR protein mutants using AF488 green fluorescence, and CALR mutant samples detected significant green fluorescence positive signals.

[0158] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An anti-CALR mutant protein antibody, It is characterized in that The antibody comprises a framework region and a complementarity determining region; The complementary determining regions include CDR-VH1, CDR-VH2 and CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL3; The complementary determining region CDR-VH1 sequence is SEQ ID NO.1; The complementary determining region CDR-VH2 sequence is SEQ ID NO.2; The complementary determining region CDR-VH3 sequence is SEQ ID NO.3; The complementary determining region CDR-VL1 sequence is SEQ ID NO.4; The complementary determining region CDR-VL2 sequence is SEQ ID NO.5; The complementary determining region CDR-VL3 sequence is SEQ ID NO.

6.

2. The anti-CALR mutant protein antibody according to claim 1, It is characterized in that The antibody comprises heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H whose sequences are shown in SEQ ID NOs: 7-10; The sequences of the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L are shown in SEQ ID NOs: 11-14.

3. An anti-CALR mutant protein antibody according to claim 1 or 2, It is characterized in that The antibody also comprises a constant region.

4. The anti-CALR mutant protein antibody according to claim 3, It is characterized in that The constant region is selected from any one of IgG1, IgG2, IgG3, and IgG4.

5. The anti-CALR mutant protein antibody according to claim 3, It is characterized in that The species origin of the constant region is cow, sheep, rabbit, mouse, rat or human.

6. Use of the anti-CALR mutant protein antibody according to claim 1 or 2 in the preparation of a reagent for detecting thrombocythaemia and / or myelofibrosis.

7. A kit for detecting thrombocythemia and myelofibrosis, It is characterized in that Including coating antibodies and labeled antibodies; The coating antibody is the monoclonal antibody according to claim 1 or 2; The labeled antibody is a monoclonal antibody targeting the N-terminus of the CALR protein.

8. The thrombocythemia and myelofibrosis detection kit according to claim 7, It is characterized in that The kit is any one of an immunohistochemistry kit, an enzyme-linked immunosorbent assay kit, a chemiluminescence immunoassay kit, a colloidal gold immunoassay kit, an immunofluorescence kit, and a flow cytometry kit.

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