CN108948205B - anti-IDH 1R132H monoclonal antibody and application thereof - Google Patents

anti-IDH 1R132H monoclonal antibody and application thereof Download PDF

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CN108948205B
CN108948205B CN201810768186.3A CN201810768186A CN108948205B CN 108948205 B CN108948205 B CN 108948205B CN 201810768186 A CN201810768186 A CN 201810768186A CN 108948205 B CN108948205 B CN 108948205B
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刘石芳
刘玉清
吴凡
柴睿超
张丽婷
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Beijing Jindai Biotechnology Co ltd
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Abstract

The invention discloses an anti-IDH 1R132H monoclonal antibody and application thereof. The heavy chain variable region of the anti-IDH 1R132H monoclonal antibody has the amino acid sequence shown in SEQ ID No.2, and the light chain variable region has the amino acid sequence shown in SEQ ID No. 4. The invention also discloses a hybridoma cell strain secreting the anti-IDH 1R132H monoclonal antibody, and the preservation number of the hybridoma cell strain is CGMCC No. 15783. Experiments prove that: the anti-IDH 1R132H monoclonal antibody has higher affinity than an anti-IDH 1R132H monoclonal antibody generated by H09 clone, has good specificity and high sensitivity, and can be used for detecting IDH1R132H mutation in glioma samples.

Description

anti-IDH 1R132H monoclonal antibody and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an anti-IDH 1R132H monoclonal antibody and application thereof.
Background
Glioma is the most common primary intracranial tumor in adults, and seriously affects the functions of cognition, language, movement and the like of patients. Its 5-year mortality is third in the mid-range of systemic tumors and is one of the globally refractory tumors. According to the morphological similarity of glioma cells and astrocytes or oligodendrocytes, the WHO glioma classification scheme mainly classifies adult diffuse gliomas into astrocytomas and oligodendrogliomas; meanwhile, according to the cellular heterogeneity, the cell proliferation condition, the vascular proliferation condition, the necrosis condition and the like, the glioma of each morphological subtype is further diagnosed from grade I to grade IV according to the malignancy grade. Among them, I, II grade is low-grade glioma (grade I astrocytoma occurs only in children), and III and IV grade are high-grade gliomas. Of the primary diagnosed glioma patients, more than 50% are the most malignant grade IV gliomas (i.e., glioblastoma, GBM), and the median survival of GBM patients is only about 14 months, and the best current treatment regimen only transiently prolongs the patient's survival. In addition, low grade gliomas often progress to high grade gliomas, resulting in a worsening of the disease. The rate of progression of different low grade gliomas varies widely, with the mechanism still unclear.
Isocitrate Dehydrogenase (IDH) is a key enzyme of the tricarboxylic acid cycle and is mainly classified into subtypes IDH1, IDH2 and IDH 3. The research shows that the mutation of IDH1(R132H) (the mutation of arginine to histidine at position 132 of IDH1, hereinafter abbreviated as IDH1 mutation) is the main marker of partial glioma. Gliomas containing the IDH1 mutation are substantially different from gliomas without the IDH1 mutation in their cytological and genetic origins and clinical phenotypes, whether they are high grade iii, iv or low grade II gliomas. Gliomas containing the IDH1 mutation may originate from oligodendrocyte progenitor cells, and about 40% of IDH1 mutant gliomas carry a co-deletion of chromosome 1p19q, with median age at about 40 years of age in onset and survival times of more than 10 years. The IDH1 wild type glioma probably originates from neural stem cells, carries 10 chromosome loss accompanied with 7 chromosome amplification, and has the onset age of more than 50 years and the median survival time of less than 15 months. Therefore, the 2016 WHO guidelines for glioma diagnosis define the IDH1 mutation as an important marker for glioma diagnosis and treatment.
IDH1 mutations in glioma samples can be detected by Sanger sequencing, pyrosequencing or NGS sequencing, but sequencing-based detection protocols do not bind to the morphology of tumors, whereas glioma samples contain normal brain tissue to varying degrees, and therefore sequencing results in a fraction of samples may be false negative results. And the antibody-based immunohistochemical detection can be tightly combined with morphology, and can identify normal brain tissues and glioma tissues, so that a false negative result in a sequencing experiment is avoided. The german Dianova company first established a monoclonal antibody (clone H09) against the IDH1 mutation, demonstrating the feasibility of monoclonal antibodies in diagnosing IDH1 mutations. Although the unit has applied for the monoclonal antibody aiming at IDH1 mutation in China, no patent and clinical permission product is available.
Disclosure of Invention
An object of the present invention is to provide an anti-IDH 1R132H monoclonal antibody.
The anti-IDH 1R132H monoclonal antibody provided by the invention comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises three complementarity determining regions CDRH1, CDRH2, CDRH 3; the light chain variable region comprises three complementarity determining regions CDRL1, CDRL2, CDRL 3;
the amino acid sequence of the CDRH1 is (a1) or (a2) or (a3) as follows:
(a1) amino acid sequence shown in 50 th to 54 th positions of SEQ ID No. 2;
(a2) amino acid sequences with the same functions are obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequences shown in the 50 th to the 54 th positions of the SEQ ID No. 2;
(a3) an amino acid sequence which has 75 percent or more than 75 percent of homology with the amino acid sequence shown in the 50 th to 54 th sites of SEQ ID No.2 and has the same function;
the amino acid sequence of the CDRH2 is (a4) or (a5) or (a6) as follows:
(a4) an amino acid sequence shown in 69 th to 85 th positions of SEQ ID No. 2;
(a5) amino acid sequences with the same function are obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequences shown in the 69 th to 85 th positions of SEQ ID No. 2;
(a6) an amino acid sequence which has 75 percent or more than 75 percent of homology with the amino acid sequence shown in the 69 th to 85 th positions of SEQ ID No.2 and has the same function;
the amino acid sequence of the CDRH3 is (a7) or (a8) or (a9) as follows:
(a7) the amino acid sequence as shown in positions 118-122 of SEQ ID No. 2;
(a8) an amino acid sequence with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the 118-122 position of the SEQ ID No. 2;
(a9) an amino acid sequence which has 75 percent or more than 75 percent of homology with the amino acid sequence shown in the 118-122 site of the SEQ ID No.2 and has the same function;
the amino acid sequence of the CDRL1 is as follows (b1) or (b2) or (b 3):
(b1) an amino acid sequence shown in the 43 th to 58 th positions of SEQ ID No. 4;
(b2) amino acid sequences with the same functions are obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequences shown in the 43 th to the 58 th positions of the SEQ ID No. 4;
(b3) an amino acid sequence which has 75 percent or more than 75 percent of homology with the amino acid sequence shown in the 43 th to 58 th positions of SEQ ID No.4 and has the same function;
the amino acid sequence of the CDRL2 is as follows (b4) or (b5) or (b 6):
(b4) amino acid sequence shown in 74 th to 80 th positions of SEQ ID No. 4;
(b5) amino acid sequences with the same functions are obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequences shown in the 74 th to the 80 th positions of SEQ ID No. 4;
(b6) an amino acid sequence which has 75 percent or more than 75 percent of homology with the amino acid sequence shown in the 74 th to 80 th sites of SEQ ID No.4 and has the same function;
the amino acid sequence of the CDRL3 is as follows (b7) or (b8) or (b 9):
(b7) the amino acid sequence as shown in position 113-121 of SEQ ID No. 4;
(b8) an amino acid sequence with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the 113 st-121 nd position of SEQ ID No. 4;
(b9) an amino acid sequence which has 75 percent or more than 75 percent of homology with the amino acid sequence shown in the 113 st-121 st position of SEQ ID No.4 and has the same function.
In the above antibody, the amino acid sequence of the heavy chain variable region is (c1) or (c2) or (c 3):
(c1) an amino acid sequence shown as SEQ ID No. 2;
(c2) an amino acid sequence with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2;
(c3) an amino acid sequence which has 75 percent or more than 75 percent of homology with the amino acid sequence shown in SEQ ID No.2 and has the same function;
the amino acid sequence of the light chain variable region is as follows (d1), (d2) or (d 3):
(d1) an amino acid sequence shown as SEQ ID No. 4;
(d2) an amino acid sequence with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 4;
(d3) and the amino acid sequence has 75 percent or more than 75 percent of homology with the amino acid sequence shown in SEQ ID No.4 and has the same function.
Further, the gene sequence encoding the heavy chain variable region is as follows (e1) or (e2) or (e 3):
(e1) DNA molecule shown in SEQ ID No. 1;
(e2) a DNA molecule having 75% or more identity to the nucleotide sequence defined in (e1) and encoding the heavy chain variable region of the antibody;
(e3) a DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in (e1) or (e2) and encodes the heavy chain variable region of the antibody;
the sequence of the encoding gene of the light chain variable region is as follows (f1), (f2) or (f 3):
(f1) a DNA molecule shown as SEQ ID No. 3;
(f2) a DNA molecule having 75% or more identity to the nucleotide sequence defined in (f1) and encoding the variable region of the light chain of the antibody;
(f3) a DNA molecule which hybridizes with the nucleotide sequence defined in (f1) or (f2) under stringent conditions and encodes the variable region of the light chain of the above-mentioned antibody.
The invention also aims to provide a hybridoma cell line 10G5-1 secreting the monoclonal antibody.
The preservation number of the hybridoma cell strain 10G5-1 secreting the monoclonal antibody provided by the invention is CGMCCNo.15783.
The hybridoma cell strain 10G5-1 is classified and named as a hybridoma cell strain, and is preserved in China general microbiological culture Collection center (CGMCC for short, the address: No.3 of West Lu No.1 of the Beijing Ind region, the institute of microbiology of the Chinese academy of sciences, zip code 100101) in 5 months and 17 days in 2018, and the preservation number is CGMCC No. 15783.
It is still another object of the present invention to provide a biomaterial related to the above antibody.
The biomaterial related to the antibody provided by the invention is any one of the following (1) to (3):
(1) nucleic acid molecules encoding the above antibodies or variable regions thereof;
(2) a vector comprising the nucleic acid molecule of (1);
(3) a host cell comprising the nucleic acid molecule of (1) or the vector of (2).
In the above biological material, the nucleic acid molecule is any one of the following (g1) to (g 3):
(g1) a DNA molecule shown as SEQ ID No.1 or SEQ ID No. 3;
(g2) a DNA molecule having 75% or more identity to the nucleotide sequence defined in (g1) and encoding the above-mentioned antibody or a variable region thereof;
(g3) a DNA molecule which hybridizes with the nucleotide sequence defined in (g1) or (g2) under stringent conditions and encodes the above-mentioned antibody or a variable region thereof.
It is a further object of the present invention to provide derivatives of the above antibodies.
The derivative provided by the invention is any one of the following (h1) - (h 6):
(h1) a fusion protein comprising the above antibody;
(h2) a multispecific or multifunctional molecule comprising the above antibody or antigen-binding portion thereof;
(h3) compositions comprising the above antibodies or antigen-binding portions thereof;
(h4) immunoconjugates comprising the above antibodies or antigen-binding portions thereof;
(h5) an antibody obtained by modifying and/or modifying the above antibody or an antigen-binding portion thereof;
(h6) an antibody comprising the above-described complementarity determining region.
It is still another object of the present invention to provide an anti-IDH 1R132H monoclonal antibody.
The anti-IDH 1R132H monoclonal antibody provided by the invention is secreted by a hybridoma cell strain with the preservation number of CGMCC No. 15783.
Still another objective of the present invention is to provide a kit for detecting or assisting in detecting whether a sample to be detected carries the IDH1R132H mutation.
The kit for detecting or assisting in detecting whether a sample to be detected carries IDH1R132H mutation or not, provided by the invention, comprises the anti-IDH 1R132H monoclonal antibody.
The invention finally provides a new use of the antibody or the hybridoma cell strain or the biological material or the derivative or the kit.
The invention provides application of the antibody or the hybridoma cell strain or the biological material or the derivative or the kit in detecting whether a sample to be detected carries IDH1R132H mutation.
The invention also provides application of the antibody or the hybridoma cell strain or the biological material or the derivative or the kit in preparing a product for detecting whether a sample to be detected carries IDH1R132H mutation.
The invention also provides the application of the antibody or the hybridoma cell strain or the biological material or the derivative or the kit in diagnosis or auxiliary diagnosis of glioma.
The invention also provides the application of the antibody or the hybridoma cell strain or the biological material or the derivative or the kit in preparing products for diagnosing or assisting in diagnosing glioma.
The invention provides an anti-IDH 1R132H monoclonal antibody, and the specificity and sensitivity of the monoclonal antibody are detected in a glioma sample. Experiments prove that: the anti-IDH 1R132H monoclonal antibody has good specificity and high sensitivity, and can be used for detecting IDH1R132H mutation in glioma samples.
Drawings
FIG. 1 is a comparison of the results of affinity assays for the 10G5-1 antibody and the H09 antibody against the R132H mutant IDH1 antigen.
FIG. 2 shows the results of immunohistochemical staining of five IDH1R132H pyrosequencing mutant samples using 10G5-1 antibody.
FIG. 3 shows the results of immunohistochemical staining of five IDH1R132H Pyrophosphoric sequencing wild-type samples with 1OG5-1 antibody.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the quantitative tests in the following examples, three replicates were set up and the results averaged.
Example 1 preparation of anti-IDH 1R132H monoclonal antibody
Preparation of antigen polypeptide
The amino acid sequence of the IDH1 wild-type antigen polypeptide is as follows: KPIIIGRHAYGDQYC. The amino acid sequence of the IDH1R132H mutant antigen polypeptide of the invention is as follows: KPIIIGHHAYGDQYC (No: U6184CB080-D), manufactured by Nanjing Kinshire Biotech, using a chemical method, wherein H is a mutation site in the antigen polypeptide sequence.
Second, animal immunization
And (3) coupling the IDH1R132H mutant antigen polypeptide prepared in the step one with KLH and then immunizing BALB/c mice. The method comprises the following specific steps:
1. mixing the IDH1R132H mutant antigen polypeptide obtained in the step one with Freund's complete adjuvant according to the ratio of 1: 1 to a concentration of 0.5 mg/ml, to obtain an antigen-polypeptide solution.
2. Basic immunity: and (3) coupling the antigen polypeptide solution obtained in the step one with KLH carrier protein to obtain KLH coupled polypeptide. KLH-conjugated polypeptide was injected intramuscularly in the extremities into BALB/c healthy mice at a dose of 50 microgram per mouse.
3. And (3) boosting immunity: after the basal immunization, booster immunization was performed in the second, fourth and seventh weeks, respectively, and the KLH-conjugated polypeptide was injected into BALB/c mice in an intramuscular injection manner at the extremity, at a dose of 25. mu.g per mouse.
Cell fusion and clone screening
Starting from the 2 nd booster immunization, blood was collected from the orbital region of mice on the 7 th day after each immunization, antibody titer was measured by ELISA, and mouse sera which had a high titer immune response against the IDH1R132H mutant antigen polypeptide (KPIIIGHHAYGDQYC) and at the same time had a relatively low titer immune response against the IDH1 wild-type polypeptide (KPIIIGRHAYGDQYC) were screened and their spleen cells were taken and fused with the mouse plasmacytoma SP2/0 cell line. After fusion, the cells are cultured on 15 culture plates with 96 wells, viable cells are screened by HAT culture medium and expanded to culture plates with 24 wells, culture supernatants of the culture plates are taken, differential immunoreaction of IDH1R132H mutant antigen polypeptide (KPIIIGHHAYGDQYC) and IDH1 wild-type polypeptide (KPIIIGRHAYGDQYC) is detected by ELISA, and 20 parent clones which can perform specific immunoreaction on of IDH1R132H mutant antigen polypeptide are found. The supernatants of the 20 parent clones were further tested in paraffin sections of glioma specimens carrying the IDH1R132H mutation and not carrying the IDH1R132H mutation, and finally 5 strains were selected for subclone culture. And verifying the subcloned culture supernatant in glioma sample paraffin sections carrying IDH1R132H mutation and not carrying IDH1R132H mutation again to finally obtain a monoclonal hybridoma cell strain 10G5-1 which can stably secrete IDH1R132H monoclonal antibody and can detect IDH1R132H mutation in glioma sample paraffin sections with high sensitivity and specificity, and recording the IDH1R132H monoclonal antibody secreted by the hybridoma cell strain 10G5-1 as the 10G5-1 monoclonal antibody.
The hybridoma cell strain 10G5-1 is classified and named as hybridoma cell strain, which has been preserved in China general microbiological culture Collection center (CGMCC, address: No.3 of the institute of microbiology of the national academy of sciences, Japan, No. 100101) of China Committee for culture Collection management of microorganisms in 5 months and 17 days in 2018, and the preservation number is CGMCC No. 15783.
The method for measuring the antibody titer by ELISA described above is as follows:
1. antigen coating: the mutant and wild type IDH1 antigen polypeptides were diluted with coating buffer as 1
Mu.g/ml, 100. mu.L per well were coated in 384-well plates and left overnight at 4 ℃;
2. washing: the next day, the liquid in the holes is poured out, and the washing solution is washed for 3 times;
3. and (3) sealing: adding 100 μ L of sealing solution into each well, and standing at room temperature for 0.5 h;
4. washing: washing with washing solution for 3 times;
5. adding the sample to be tested (primary antibody): sera containing monoclonal antibodies were serially diluted in PBS on another plate (serial dilution from 1: 1000 to 1: 512000 according to a 1: 2 gradient) and added to the coated plate in 100. mu.L per well in duplicate, with mouse sera before immunization as a negative control. Incubate 1-2h in a 37 ℃ incubator with a lid.
6. Washing: washing with washing solution for 3 times;
7. adding an enzyme-labeled secondary antibody (Peroxidase-conjugated affinity Goat Anti-rabbitIgG, FC gamma Fragment Specific), and adding a blocking solution according to the ratio of l: diluting at the ratio of 1000, adding 100 mu L of the diluted solution into each hole, and covering a constant temperature box at 37 ℃ for incubation for 1 h;
8. washing: washing with washing solution for 5 times, and washing with distilled water for 2 times;
9. color development: adding a freshly prepared substrate solution, keeping the substrate solution at 100 mu L/hole, and standing the substrate solution at room temperature in a dark place for 5-30 min until the substrate solution shows blue;
10. terminating reaction and carrying out color comparison: add 50. mu.L of stop buffer to each well. The color turns yellow; the absorbance of each well at 450nm was measured using a microplate reader.
Preparation and purification of tetra, 10G5-1 monoclonal antibody
Amplifying the hybridoma cell line 10G5-1 obtained in the step three in a DMEM medium containing 10% (volume fraction) FBS, culturing in a rotary flask, removing the precipitate after culturing for 14 days, and taking the supernatant; the supernatant was centrifuged to remove cell debris, concentrated, and subjected to a standardized protein A purification protocol to obtain 18mg of 90% pure 10G5-1 monoclonal antibody solution.
Five, 10G5-1 monoclonal antibody sequence
The 10G5-1 monoclonal antibody was sequenced. The method comprises the following specific steps: separating total RNA from the hybridoma cell strain 10G5-1, and performing reverse transcription to obtain cDNA; and carrying out PCR amplification by using the cDNA as a template to obtain VH and VL sequences of the antibody. The amplified sequence is then recombined into a plasmid and transformed into E.coli. Colony PCR was then used to screen bacterial clones with the correct insert length. From these five clones with the correct insert length were selected and DNA extracted for sequencing. Comparing the sequences from different clones, the identical sequence in these clones is the nucleotide sequence corresponding to the antibody, and determining the amino acid sequence of the corresponding protein according to the nucleotide sequence.
The sequencing result shows that: the amino acid sequence of the heavy chain variable region of the 10G5-1 monoclonal antibody is shown as SEQ ID No.2, wherein the amino acid sequence of the framework region FR1 is 20-49 th of SEQ ID No.2, the amino acid sequence of the complementary determining region CDRH1 is 50-54 th of SEQ ID No.2, the amino acid sequence of the framework region FR2 is 55-68 th of SEQ ID No.2, the amino acid sequence of the complementary determining region CDRH2 is 69-85 th of SEQ ID No.2, the amino acid sequence of the framework region FR3 is 86-117 th of SEQ ID No.2, the amino acid sequence of the complementary determining region CDRH3 is 118-122 th of SEQ ID No.2, and the amino acid sequence of the framework region FR4 is 123-133 th of SEQ ID No. 2. The coding gene sequence of the heavy chain variable region of the 10G5-1 monoclonal antibody is shown in SEQ ID No. 1.
The amino acid sequence of the light chain variable region of the 10G5-1 monoclonal antibody is shown as SEQ ID No.4, wherein the amino acid sequence of the framework region FR1 is 20-42 th of SEQ ID No.4, the amino acid sequence of the complementary determining region CDRL1 is 43-58 th of SEQ ID No.4, the amino acid sequence of the framework region FR2 is 59-73 th of SEQ ID No.4, the amino acid sequence of the complementary determining region CDRL2 is 74-80 th of SEQ ID No.4, the amino acid sequence of the framework region FR3 is 81-112 th of SEQ ID No.4, the amino acid sequence of the complementary determining region CDRL3 is 113-121 th of SEQ ID No.4, and the amino acid sequence of the framework region FR4 is 122-131 th of SEQ ID No. 4. The coding gene sequence of the heavy chain variable region of the 10G5-1 monoclonal antibody is shown in SEQ ID No. 3.
Example 2 affinity assay for anti-IDH 1R132H monoclonal antibody
The affinity of the anti-IDH 1R132H monoclonal antibody produced by the hybridoma cell line 10G5-1 of the present invention and the affinity of the anti-IDH 1R132H monoclonal antibody produced by the currently available H09 clone to the IDH1R132H mutant antigen polypeptide were determined and compared using the Biacore 8k platform. The method comprises the following specific steps:
1. the IDH1R132H mutant antigen polypeptide was immobilized on the substrate at 25 ℃ and the working buffer was HBS-EP buffer. The fixing process is as follows: first, flow cells of a CM-5 sensor chip were activated for 100s with 50mmol/L NHS (N-hydroxysuccinimide) and 200mmol/L EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) at a flow rate of 10. mu.L/min. Subsequently, the antigen polypeptide was diluted to 10mmol/L with sodium acetate (pH5.0) at a concentration of 1. mu.g/mL. And a second flow cell is injected to a level of 5 units of response while the second flow cell remains blank. When the amino coupling is complete, the remaining coupling sites are blocked for 200s with a 1mol/L solution of ethanolamine hydrochloride.
2. The antibodies to be detected are diluted in the following gradient: 10G 5-1: 0nM, 1.25nM, 2.5nM, 5nM, 10nM, 20nM, 40 nM; HA 09: 0nM, 6.25nM, 12.5nM, 25nM, 50nM, 100nM, 200nM, and then detected by flowing through the flow cell at 30. mu.L/min, respectively. When one antibody was detected, the immobilized polypeptide was rapidly eluted using glycine-hydrochloric acid solution of pH1.5 to regenerate the flow cell.
3. The results of the computational experiments were evaluated using Biacore 8k evaluation software.
The results are shown in Table 1. The results show that: the affinity of the anti-IDH 1R132H monoclonal antibody generated by the hybridoma cell strain 10G5-1 to IDH1R132H mutant antigen polypeptide (U6184CB080-D) is higher than that of the anti-IDH 1R132H monoclonal antibody generated by the current general H09 clone, and the affinity equilibrium constant of the anti-IDH 1R132H monoclonal antibody is nearly 10 times lower than that of H09.
TABLE 1 comparison of affinity assay results
Figure BDA0001729564120000081
Example 3 application of anti-IDH 1R132H monoclonal antibody and verification of sensitivity and specificity thereof
Five glioma tissue samples carrying IDH1R132H mutation and five glioma tissue samples not carrying IDH1R132H mutation (provided by Beijing neurosurgery research) verified by pyrosequencing are used as samples to be tested to carry out immunohistochemical experiments, and the sensitivity and specificity of the anti-IDH 1R132H monoclonal antibody generated by the hybridoma cell strain 10G5-1 are detected. The method comprises the following specific steps: 1. baking slices: placing the glioma paraffin slices into a baking piece at 60 ℃ for one night; 2. dewaxing and hydrating: sequentially soaking slices with xylene I (10min), xylene II (10min), absolute ethyl alcohol I (5min), absolute ethyl alcohol II (5min), 95% ethyl alcohol I (5min), 95% ethyl alcohol II (5min), 75% ethyl alcohol I (5min) and 75% ethyl alcohol II (5min), finally washing with distilled water for 1 min and 1 XPBS for 5 min; 3. antigen retrieval: the slices were completely immersed in a beaker (pH 8.0) containing EDTA antigen retrieval solution, boiled in water in an autoclave, and then the beaker was placed in the autoclave and the lid was closed. Heating until the valve starts to rotate, timing for three minutes, stopping heating, naturally cooling to room temperature, and washing the slices with PBS (phosphate buffer solution) for 3 minutes and 3 times; 4. blocking endogenous peroxidase: adding a proper amount of 3% hydrogen peroxide dropwise, incubating for 10 minutes at room temperature, and washing the slices for 3 minutes by 3 times by using PBS (phosphate buffer solution); 5. primary antibody incubation: dropping 100 microliter primary antibody, placing in a wet box, standing overnight at 4 ℃, washing the slices with PBS buffer solution for 3 minutes and 3 times; 6. and (3) sealing: dropping 100 microliter goat serum, sealing at room temperature for 30min, and washing the slices with PBS buffer solution for 3 min × 3 times; 7. and (3) secondary antibody incubation: dripping a proper amount of reaction enhancing solution, and incubating for 30 minutes at room temperature; washing with PBS buffer solution for 3 min × 3 times, then adding dropwise the reinforced enzyme goat anti-mouse IGG polymer, incubating for 30min at room temperature, washing slices with PBS buffer solution for 3 min × 3 times; 8. color development: dropwise adding a DAB color developing solution which is prepared freshly for color development for 2 minutes, and performing a reaction in distilled water; 9. counterdyeing: bleaching and dyeing for 8 seconds by using hematoxylin staining solution, and quickly cutting off the staining by using distilled water; 10. dehydrated, transparent, and then encapsulated with neutral gum, and after drying, the staining was observed under a microscope.
Results as shown in fig. 2 and 3, fig. 2 shows the results of immunohistochemical staining of five IDH1R132 pyrosequencing mutant samples, and fig. 3 shows the results of immunohistochemical staining of five IDH1R132 pyrosequencing wild-type samples. The results show that: the 10G5-1 monoclonal antibody showed positive results in five paraffin sections of glioma samples with the IDH1R132H mutation, and negative results in five paraffin sections of glioma samples without the IDH1R132H mutation. The antibody of the invention is shown to have higher sensitivity and specificity.
Sequence listing
<110> Beijing Dajin bioscience, Inc
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Claims (10)

1. An anti-IDH 1R132H monoclonal antibody, said antibody comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises three complementarity determining regions CDRH1, CDRH2, CDRH 3; the light chain variable region comprises three complementarity determining regions CDRL1, CDRL2, CDRL 3;
the amino acid sequence of the CDRH1 is shown as the 50 th to 54 th positions of SEQ ID No. 2;
the amino acid sequence of the CDRH2 is shown as the 69 th to 85 th positions of SEQ ID No. 2;
the amino acid sequence of the CDRH3 is shown as the 118-122 position of SEQ ID No. 2;
the amino acid sequence of the CDRL1 is shown as the 43 th to 58 th positions of SEQ ID No. 4;
the amino acid sequence of the CDRL2 is shown as the 74 th to the 80 th positions of SEQ ID No. 4;
the amino acid sequence of the CDRL3 is shown as the position 113-121 of SEQ ID No. 4.
2. The monoclonal antibody of claim 1, characterized in that:
the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 2;
the amino acid sequence of the light chain variable region is shown as SEQ ID No. 4.
3. The monoclonal antibody of claim 2, characterized in that:
the coding gene sequence of the heavy chain variable region is a DNA molecule shown in SEQ ID No. 1;
the coding gene sequence of the light chain variable region is a DNA molecule shown in SEQ ID No. 3.
4. A hybridoma cell line 10G5-1 secreting the monoclonal antibody of any one of claims 1 to 3, with the preservation number of CGMCC No. 15783.
5. The biomaterial-associated monoclonal antibody according to any one of claims 1 to 3, which is any one of the following (1) to (3):
(1) a nucleic acid molecule encoding the antibody or variable region thereof of any one of claims 1-3;
(2) a vector comprising the nucleic acid molecule of (1);
(3) a host cell comprising the nucleic acid molecule of (1) or the vector of (2).
6. The biomaterial of claim 5, wherein: the nucleic acid molecule is a DNA molecule shown in SEQ ID No.1 or SEQ ID No. 3.
7. The derivative of the monoclonal antibody according to any one of claims 1 to 3, which is any one of the following (h1) to (h 6):
(h1) a fusion protein comprising the monoclonal antibody of any one of claims 1-3;
(h2) a multispecific or multifunctional molecule comprising a monoclonal antibody or antigen-binding portion thereof according to any one of claims 1-3;
(h3) a composition comprising the monoclonal antibody or antigen-binding portion thereof of any one of claims 1-3;
(h4) an immunoconjugate comprising the monoclonal antibody or antigen binding portion thereof of any one of claims 1-3;
(h5) an antibody obtained by modifying and/or engineering the monoclonal antibody or the antigen-binding portion thereof according to any one of claims 1 to 3;
(h6) an antibody comprising the complementarity determining region of any one of claims 1-3.
8. An anti-IDH 1R132H monoclonal antibody secreted by hybridoma cell strain with preservation number of CGMCC No. 15783.
9. A kit for detecting or aiding in the detection of whether a test sample carries an IDH1R132H mutation, comprising the monoclonal antibody of any one of claims 1-3 or the monoclonal antibody of claim 8.
10. Use of the monoclonal antibody of any one of claims 1-3 or the hybridoma cell line 10G5-1 of claim 4 or the biological material of claim 5 or 6 or the derivative of claim 7 or the monoclonal antibody of claim 8 or the kit of claim 9 for preparing a product for detecting whether a test sample carries the IDH1R132H mutation;
or, the monoclonal antibody of any one of claims 1-3 or the hybridoma cell line 10G5-1 of claim 4 or the biological material of claim 5 or 6 or the derivative of claim 7 or the monoclonal antibody of claim 8 or the kit of claim 9, for use in the preparation of a product for diagnosing or aiding in the diagnosis of glioma.
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CN106957367A (en) * 2017-03-31 2017-07-18 北京爱仁医疗科技有限公司 Anti- IDH1 R132H antibody and its production and use
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CN107151269A (en) * 2016-03-04 2017-09-12 四川科伦博泰生物医药股份有限公司 A kind of antibody of PDL 1, its medical composition and its use
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