CN117003866A - An anti-CALR mutant protein antibody, preparation method and detection kit - Google Patents

Abstract

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

Description

An anti-CALR mutant protein antibody, preparation method and detection kit

Technical field

The present invention relates to the technical fields of antibody preparation and immunological detection, and specifically relates to an anti-CALR (myeloproliferative tumor marker calcium reticulum) mutant protein antibody, preparation method and detection kit

Background technique

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

In recent years, mutations in multiple molecular markers such as JAK2, MPL, and TET have been discovered one after another. The discovery of these markers is of great significance for the detection and diagnosis of MPN. However, studies have shown that 30%-45% of patients with ET or MF who carry wild-type JAK2/MPL still have diagnostic difficulties.

In recent years, multiple studies have shown that characteristic calreticulin (CALR) mutations are found in MPN patients who are negative for JAK2 mutations, suggesting that calreticulin (CALR) mutations are expected to become a new diagnostic tool for myeloproliferative tumors. Molecular markers.

Current research shows that there are more than 50 types of mutations in CALR. The most common mutation types are the deletion of 52 bases on 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 this type II mutation accounts for approximately 80% of the patients carrying all CALR mutations. Although there are various deletion or insertion mutations in CALR exon 9, no matter what the mutation form, it will lead to a one base pair reading frame shift, thereby producing a gene with a lack of endoplasmic reticulum retention sequence (KDEL amino acid sequence) of a novel C-carboxyl-terminal protein. The detection of this CALR mutant peptide may become a new idea for clinical detection of CALR mutations.

At present, studies have prepared polyclonal antibodies and monoclonal antibodies (CAL2) based on the characteristics of the CALR mutant protein for immunohistochemical staining of bone marrow tissue in MPN patients. The polyclonal antibodies have non-specific staining. Although CAL2 is a monoclonal antibody, Cloned antibodies can only be used for immunohistochemical detection of formalin-fixed bone marrow wax blocks. The result analysis relies heavily on the subjective judgment of technicians, making it impossible to achieve standardized detection of markers and disease diagnosis.

Contents of the invention

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

As an improvement to the anti-CALR mutant protein antibody, the amino acid sequence of the heavy chain complementarity determining region of the antibody is SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or the sequence is replaced or deleted. Or an amino acid sequence with equivalent functions formed by adding one or more amino acids; the amino acid sequence of the light chain complementarity determining region is SEQ ID NO.4, SEQID NO.5, SEQ ID NO.6 or the sequence is replaced, deleted or added A functionally equivalent amino acid sequence formed by one or more amino acids.

As a further improvement to the anti-CALR mutant protein antibody, the antibody includes the heavy chain backbone regions FR1-H, FR2-H, FR3-H and FR4-H whose sequences are shown in SEQ ID NO: 7-10; The sequences are the light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L shown in SEQ ID NO: 11-14 in sequence.

As a further improvement to the anti-CALR mutant protein antibody, the antibody further includes a constant region.

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

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

A method for preparing an anti-CALR mutant protein antibody, including the following steps: S1, based on the abnormal amino acid segment of the C-carboxyl terminus of the CALR mutant protein, design and synthesize a dominant antigen epitope polypeptide, and couple the polypeptide to a carrier protein to form a complete antigen; S2 Mix the complete antigen prepared in the previous step with Freund's complete adjuvant in equal volumes and emulsify evenly; after multiple immunizations of Balb/c mice, collect blood from the orbit of each mouse and centrifuge to separate the serum; measure the serum titer in S3 and take the serum potency. Mice with a price greater than 10 ⁷ were subjected to intraspleen boosting immunization; after S4 boosting immunization, spleen cells were fused with sp2/0 myeloma cells in proportion; after S5 fusion, culture medium was added to terminate the fusion, and the cells were resuspended in culture medium and placed Cultivate in an incubator; after S6 fusion cell culture, detect specific antibodies in the cell culture supernatant to obtain hybridoma cells; S7 take healthy mice, intraperitoneally inject paraffin and then inject hybridoma cells respectively, collect ascites, and obtain monoclonal Antibody.

Application of an anti-CALR mutant protein antibody in preparing a reagent for detecting thrombocythemia and/or myelofibrosis.

A kit for detecting thrombocythemia and myelofibrosis, including a coating antibody and a labeled antibody; the coating antibody is a monoclonal antibody as claimed in claim 1, 2 or 3; the labeled antibody is for Monoclonal antibody to the N-terminus of CALR protein.

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

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

The novel anti-CALR mutant protein monoclonal antibody provided in this application has a new antigen-binding domain, which only specifically binds to the CALR mutant protein and does not bind to the wild-type CALR protein. It has strong activity and affinity, high stability, etc. advantage.

The above-mentioned monoclonal antibodies and kits have strong specificity and high sensitivity when used to detect serum CALR mutant proteins, with a detection range of 2.93-375ng/mL.

The above-mentioned detection reagents can effectively promote the application of CALR mutant protein detection, promote the inclusion of CALR mutant protein detection in routine clinical testing, and play an important role in the clinical diagnosis of thrombocythemia and myelofibrosis.

Description of the drawings

In order to more clearly explain the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly introduced below:

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

Figure 2 Immunohistochemical staining diagram of monoclonal antibody against CALR mutant protein;

Figure 3 CALR mutant protease-linked immunosorbent (ELISA) kit standard curve;

Figure 4 Example of enzyme-linked immunosorbent (ELISA) kit for detecting the concentration of CALR mutant protein in 20 clinical serum samples;

Figure 5 Schematic diagram of the interpretation method of CALR mutant cell standards using a flow cytometry kit.

Detailed ways

In order to facilitate understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. Typical embodiments of the invention are shown in the drawings.

Example 1 Preparation and identification of monoclonal antibodies against CALR mutant protein

1.1 Immunogen design and preparation

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

The glutaraldehyde method was used to couple the peptide with keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) to form complete antigens (CALR-1-KLH, CALR-2-KLH, CALR-1-BSA, CALR- 2-BSA).

1.2 Mouse immunization

Mix the CALR-1-KLH complete antigen prepared in the previous step and Freund's complete adjuvant in equal volumes and emulsify evenly;

Immunize 6-week-old Balb/c mice by subcutaneous injection at multiple points, 3 times with an interval of 2 weeks each time;

Two weeks after the third immunization, blood was collected from the orbit of each mouse, and the serum was separated by centrifugation. The serum titer was determined by indirect ELISA. Mice with a serum titer greater than 10 ⁷ were selected for intraspleen booster immunization.

1.3 Cell fusion

Three days after the booster immunization, spleen cells were fused with sp2/0 myeloma cells at a ratio of 6:1, and the fusion agent was PEG (MW1450, sigma);

After 10 minutes of fusion, add fresh serum-free medium to terminate fusion, resuspend the cells in DMEM culture medium containing 2% HAT, and culture them in a 37°C, 5% CO2 incubator.

1.4 Clone screening

Cultivate the fused cells for 7-10 days, and use a 96-well plate coated with CALR-1-BSA and CALR-2-BSA to detect specific antibodies in the cell culture supernatant using an indirect method;

If the supernatant of the same cell line reacts only with CALR-1-BSA and the result is positive, but the reaction with CALR-2-BSA is negative, then the cell line is determined to be a hybridoma cell that can secrete antibodies that specifically recognize the CALR mutant protein. strain;

After three consecutive subclonings, when the antibody positive rate was 100% detected by ELISA, it was determined to be hybridoma cells stably expressing the target antibody. Finally, three hybridoma cells with strong specificity and high titer were obtained, named Cal26B10, Cal26B11, Cal26B15, and preserved.

1.5 Preparation and purification of mouse monoclonal antibodies

Healthy F1 mice were taken and intraperitoneally injected with paraffin, 0.5ml/mouse. Seven days later, Cal26B10, Cal26B11, and Cal26B15 cells were injected at a dose of ¹⁰ cells/mouse. Ascites was collected after 7-10 days, and purified through octanoic acid-ammonium sulfate and Protein. A. Affinity purification to obtain mouse monoclonal antibodies with higher purity (>95%).

1.6 Identification of monoclonal specificity of anti-CALR mutant protein

Collect peripheral blood from patients with thrombocythemia (ET) and myelofibrosis (MF), isolate peripheral blood mononuclear cells with Ficoll, extract total protein, use Western blot method to detect the specificity of the antibody, and use lentivirus to stably transfer CALR wild-type plasmid The total protein of 293T cells was used as a negative control, and the total protein of 293T cells stably transfected with CALR type I mutant and CALR type II mutant plasmids was used as a positive control.

Western blot results show (as shown in Figure 1) that Cal26B10, Cal26B11, and Cal26B15 can react with CALR mutation-positive clinical peripheral blood mononuclear cell samples and 293T cell samples overexpressing mutant CALR protein, but not with overexpressing wild-type CALR. The 293T cell sample did not react with the protein, indicating that the antibody specificity was good.

In Figure 1 (preparation of antibody specificity detection results), WT: 293T cells overexpressing wild-type CALR; del52: 293T cells overexpressing CALR type I mutations; ins5: 293T cells overexpressing CALR type II mutations; P1: carrying Peripheral blood PBMC cells of MPN patients with CALR type I mutations; P2: Peripheral blood PBMCs of MPN patients with CALR type II mutations.

Example 2 Determination of monoclonal antibody affinity against CALR mutant protein

WeSPR 100 was used to determine the affinity of each antibody, and the results are shown in Table 1 (anti-CALR mutant protein monoclonal antibody affinity detection results). Cal26B11, which has the highest affinity, was selected as the monoclonal antibody against CALR mutant protein for detection and used for subsequent detection and further research and development.

Ka(M-1s-1) Kd(s-1) KD(M) Cal26B10 1.24×10 ⁵ 6.80× ^10-4 5.48× ^10-9 Cal26B11 3.00×10 ⁵ 1.77× ^10-4 5.90×10 ^-10 Cal26B15 4.48×10 ⁵ 3.36× ^10-4 7.51×10 ^-10

Table 1 Anti-CALR mutant protein monoclonal antibody affinity detection results

Example 3 Analysis of Cal26B11 monoclonal antibody variable region

3.1 Gene fishing

The monoclonal antibody Cal26B11 hybridoma cell line is expanded and cultured. After entering the logarithmic growth cycle, 10 ⁷ cells are taken, the total cellular RNA is extracted, and the cDNA product is obtained through reverse transcription. The product is amplified with rTaq DNA polymerase and After recovery, insert into the pMD-18T vector and transform into DH5α competent cells. After the colonies grow, take 3 heavy chain and light chain gene clones and send them to a gene sequencing company for sequencing.

3.2 Sequence analysis of heavy chain and light chain variable region genes

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

CAGATCCAGTTGGTGCAGTCTGGACCTGTTCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAAACTATGGAATGACCTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAAAGCCAACATATGCTGGTGACTTCAAGGGACGATTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACGACCTC AAAAATGAGGACACGGCTACATATTTCTGTTCAAGCCAATTACGAGGGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

Heavy chain variable region CDRH1 amino acid sequence:

GYTFTNY(SEQ ID NO:1)

Heavy chain variable region CDRH2 amino acid sequence:

NTYTGK(SEQ ID NO:2)

Heavy chain variable region CDRH3 amino acid sequence:

FCSSQLRGAY(SEQ ID NO:3)

Amino acid sequence of heavy chain framework region:

FR1-H：QIQLVQSGPVLKKPGETVKISCKAS(SEQ ID NO:7)

FR2-H：GMTWVKQAPGKGLKWMGWI(SEQ ID NO:8)

FR3-H：PTYAGDFKGRFAFSLETSASTAYLQINDLKNEDTATY(SEQ ID NO:9)

FR4-H：WGQGTLVTVSA(SEQ ID NO:10)

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

GACATTGTGATGTCACAGTCTCCGTCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATAGTACCAATCAGAAGAACTCCTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGG CTGAAGACCTGGCAGTTTTATTACTGTCAGCAATATTATAGCTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

Light chain variable region CDRL1 amino acid sequence:

KSSQSLLYSTNQKNSLA(SEQ ID NO:4)

Light chain variable region CDRL2 amino acid sequence:

WASTRES(SEQ ID NO:5)

Light chain variable region CDRL3 amino acid sequence:

CQQYYSYPWT(SEQ ID NO:6)

Amino acid sequence of light chain framework region:

FR1-L：DIVMSQSPSSLAVSVGEKVTMSC(SEQ ID NO:11)

FR2-L：WYQQKPGQSPKLLIY(SEQ ID NO:12)

FR3-L：GVPDRFTGSGSGTDFTLTISSVKAEDLAVYY(SEQ ID NO:13)

FR4-L: FGGGTKLEIK(SEQ ID NO:14)

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

4.1 Clinical sample collection

Bone marrow tissue wax blocks of MPN (PV, ET, MF) patients from the outpatient and inpatient wards of Huashan Hospital North Branch and Changhai Hospital from 2015 to 2017 were collected, a total of 28 cases;

Among them, 5 cases carried CALRI mutations, 4 cases carried CALRII mutations, 1 case carried CALR V (c.1091_1142del) mutations, and 18 cases carried JAK2 mutations. Cal26B11 antibody was used to immunohistochemically stain the collected clinical samples;

Immunohistochemical staining results were scored according to ASCO/CAP guidelines (American Society of Clinical Oncology/College of American Pathologists).

4.2 Immunohistochemical staining

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

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

(3) Gradient washing with ethanol to remove xylene and washing in double-distilled water for 5 minutes.

(4) Block endogenous peroxidase activity with 0.3% H ₂ O ₂ at room temperature for 10 minutes.

(5) Wash 4 times in double-distilled water, 5 minutes each time.

(6) Add Cal26B11 monoclonal antibody dropwise on the tissue sections and incubate at 4°C for 12 hours.

(7) Wash PBS buffer 4 times for 5 minutes each time.

(8) Add horseradish peroxidase-labeled secondary antibody dropwise and incubate at 37°C for 30 minutes.

(9) Wash PBS buffer 4 times, 5 minutes each time.

(10) Add DAB chromogen dropwise and incubate at room temperature to develop color.

(11) Wash with double steamed water 4 times, 5 minutes each time.

(12) Dry, seal, and observe under microscope.

It was found that the immunohistochemical staining results were completely consistent with the Sanger sequencing results, and samples with only CALR mutations were positively stained, while samples without CALR mutations were not stained (Figure 2).

Sample number clinical diagnosis Sanger sequencing test 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 —

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

Figure 2. Results of staining bone marrow sections of MPN patients using Cal26B11 antibody using immunohistochemistry (×200 times). A-I correspond to the relevant sample numbers in Table 2:

A: The corresponding sample number is P8;

B: The corresponding sample number is P9;

C: The corresponding sample number is P11;

D: The corresponding sample number is P14;

E: The corresponding sample number is P19;

F: The corresponding sample number is P20;

G: The corresponding sample number is P22;

H: The corresponding sample number is P24;

I: The corresponding sample number is P28.

Example 5 Construction of CALR mutant protein detection kit

The kit contains coated antibody Cal26B11 and labeled antibody C7492 (Sigma-Aldrich). The kit is an enzyme-linked reaction kit. The specific steps are as follows:

Dilute the coating antibody Cal26B11 with CB to 10 μg/ml, add 100 μl per well into the wells of the enzyme plate, and coat overnight at 4°C;

Pat the enzyme plate dry the next day, add 200 μl of blocking solution (PBS containing 1% BSA) to each well, block at 37°C for 2 hours, and pat dry after blocking;

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

After incubating for 1 hour at 37°C, wash the plate three times with 0.05% PBST;

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

Add 100 μl TMB chromogenic solution to each well and incubate at 37°C for 10 minutes;

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

5.1 Kit detection linearity

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

Table 3. Linear detection of paired antibodies

It can be seen from the experimental data in Table 3 and Figure 3 above that the kit obtained in this application using coated antibody Cal26B11 and labeled antibody C7492 (Sigma-Aldrich) still has obvious antigen concentration at a concentration as low as 2.93ng/mL. Positive performance shows a good linear relationship between 2.93-375ng/mL, and the fitting coefficients are all greater than 0.99. This proves that the monoclonal antibody used in this application has a lower detection limit, is highly sensitive in practical applications, and has broad application prospects in qualitative detection.

5.2 Kit detection precision

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

The CALR mutation standard protein was added to the negative serum at a certain concentration, and 3 batches of reagents were used for detection. The detection was continued for 5 days. The inter-batch difference analysis of the reagents was performed. The inter-batch CV was less than 3%, and the inter-batch precision met the requirements (Table 4 ).

Table 4 Standard concentration and corresponding absorbance (OD) value

5.3 Kit detection specificity

The kit test simultaneously detects the same concentration of CALR mutant protein standard and CALR wild-type protein standard (187.5ng/mL, 46.88ng/mL, 11.72ng/mL), and uses t test for statistical analysis, p<0.05, the results prove The established CALR mutant protein ELISA kit has good specificity (Table 5).

Table 5 Standard concentration and corresponding absorbance (OD) value

5.4 Kits for testing clinical samples

Ten sera from patients with CALR mutations (type I - J1522, J1525, J1526, J1531, J1535) and type II - J1550, J1567, J1576, J1580, J1675) and 5 sera from patients with JAK2 mutations (J1504, J1505, J1516, J1538) were selected. , J1618), 5 negative patient serum samples (P1-P5) were tested using the CALR mutant protein ELISA reagent established above. The measured concentrations of the CALR group were significantly different from those of the JAK2 group or the wild group (Table 6, Figure 4).

In Figure 4, the ELISA kit detects the serum of 10 cases of CALR mutated MPN, 5 cases of JAK2 mutated MPN, and 5 cases of normal physical examination volunteers. The CALR mutant protein concentration in peripheral serum of patients with CALR mutations confirmed by molecular detection methods is significantly higher than that of JAK2 mutations. Type MPN and normal physical examination volunteers.

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

patient number Absorbance values CALR protein concentration patient number Absorbance values 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

Table 6 Absorbance values detected in clinical samples and corresponding CALR protein concentrations

Example 6 Establishment of CALR mutant protein flow cytometry detection kit

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 steps are as follows:

Collect cells by trypsin digestion and centrifuge at 300g for 6 minutes;

Remove the supernatant, add 1 ml of PBS for washing, centrifuge at 300g for 6 minutes, and remove the supernatant;

Add 100 μl 4% paraformaldehyde to every 10 ⁶ cells for fixation and mix well;

Incubate at room temperature for 20 minutes;

Add 1 ml of PBS to wash the cells, centrifuge at 300 g for 6 minutes, remove the supernatant, and repeat once (wash the cells twice, repeat once by adding 1 ml of PBS to wash the cells, centrifuge at 300 g for 6 minutes, remove the supernatant);

Add 100 μl of pre-cooled 90% methanol to every ¹⁰ cells to break the membrane, and incubate on ice for 15 minutes;

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

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;

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

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

Add 1 ml of PBS to wash, centrifuge at 300 g for 6 minutes, remove the supernatant, and repeat once (wash the cells twice, that is, repeat once, add 1 ml of PBS to wash the cells, centrifuge at 300 g for 6 minutes, remove the supernatant).

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

Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art will understand that various changes, modifications, and substitutions can be made to these embodiments without departing from the principles and spirit of the invention. and modifications, the scope of the invention is defined by the appended claims and their equivalents.

Claims (10)

1. An anti-CALR mutein antibody characterized in that the antibody comprises a framework region and a complementarity determining region;

the complementarity determining regions include CDR-VH1, CDR-VH2 and CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL3;

the CDR-VH1 sequence of the complementarity determining region is SEQ ID NO.1;

the CDR-VH2 sequence of the complementarity determining region is SEQ ID NO.2;

the CDR-VH3 sequence of the complementarity determining region is SEQ ID NO.3;

the CDR-VL1 sequence of the complementarity determining region is SEQ ID NO.4;

the CDR-VL2 sequence of the complementarity determining region is SEQ ID NO.5;

the CDR-VL3 sequence of the complementarity determining region is SEQ ID NO.6.

2. The anti-CALR mutein antibody of claim 1, wherein the amino acid sequence of the heavy chain complementarity determining region of the antibody is SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or an amino acid sequence having equivalent function formed by substitution, deletion or addition of one or more amino acids to the sequence;

the amino acid sequence of the light chain complementary determining region is SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or an amino acid sequence with the same function formed by replacing, deleting or adding one or more amino acids.

3. The anti-CALR mutein antibody of claim 1, characterized in that the antibody comprises the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H, the sequences of which are shown in sequence SEQ ID NOs 7-10;

the sequence is shown as SEQ ID NO. 11-14, and the light chain skeleton region FR1-L, FR2-L, FR-L and FR4-L are shown in sequence.

4. An anti-CALR mutein antibody according to claim 1, 2 or 3, characterized in that the antibody further comprises a constant region.

5. The anti-CALR mutein antibody of claim 4, wherein the constant region is selected from the group consisting of a constant region of any one of IgG1, igG2, igG3, igG 4.

6. The anti-CALR mutein antibody of claim 4, wherein the constant region is of bovine, ovine, rabbit, mouse, rat or human origin.

7. A method of making an anti-CALR mutein antibody of claim 1, 2 or 3, comprising the steps of:

s1, designing and synthesizing dominant antigen epitope polypeptide according to an amino acid segment with abnormal C-carboxyl terminal of a CALR mutant protein, and coupling the polypeptide with carrier protein to form complete antigen;

s2, mixing the complete antigen prepared in the previous step with Freund' S complete adjuvant in equal volume and emulsifying uniformly; after multiple immunizations of Balb/c mice, each mouse was collected from the orbit and serum was isolated by centrifugation;

s3, measuring serum titer, and taking the serum titer to be more than 10 ⁷ Is subjected to spleen internal boosting immunization;

s4, taking spleen cells and sp2/0 myeloma cells to fuse in proportion after enhancing immunity;

s5, adding a culture medium to terminate fusion after fusion, and resuspending cells with a culture solution, and culturing in an incubator;

s6, after the fusion cell culture, detecting a specific antibody in a cell culture supernatant to obtain a hybridoma cell;

s7, taking a healthy mouse, injecting paraffin into the abdominal cavity, then respectively injecting hybridoma cells, and collecting ascites to obtain the monoclonal antibody.

8. Use of the anti-CALR mutein antibody according to claim 1, 2 or 3 for the preparation of a reagent for the detection of thrombocythemia and/or myelofibrosis.

9. A kit for the detection of thrombocythemia and myelofibrosis, comprising a coated antibody and a labeled antibody;

the coated antibody is the monoclonal antibody of claim 1, 2 or 3;

the labeled antibody is a monoclonal antibody aiming at the N end of the CALR protein.

10. The kit for detecting thrombocythemia and myelofibrosis according to claim 9, wherein the kit is any one of an immunohistochemical kit, an enzyme-linked immunosorbent kit, a chemiluminescent immunosorbent kit, a colloidal gold immunosorbent kit, an immunofluorescent kit, and a flow cytometry kit.