CN113186284A - Nucleic acid-antibody dual cancer detection kit - Google Patents
Nucleic acid-antibody dual cancer detection kit Download PDFInfo
- Publication number
- CN113186284A CN113186284A CN202110487543.0A CN202110487543A CN113186284A CN 113186284 A CN113186284 A CN 113186284A CN 202110487543 A CN202110487543 A CN 202110487543A CN 113186284 A CN113186284 A CN 113186284A
- Authority
- CN
- China
- Prior art keywords
- seq
- cxcl
- antibody
- cxcl1
- chain variable
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/521—Chemokines
- G01N2333/522—Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4 or KC
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a nucleic acid-antibody dual cancer detection kit. The invention obtains the specific RPA primer and probe aiming at CXCL1 detection by analyzing the CXCL1 gene sequence; meanwhile, monoclonal antibodies with good effects are screened and obtained aiming at the CXCL1 protein, and the monoclonal antibodies are used for preparing an immunodetection kit. The two detection methods are combined for use, so that the detection accuracy can be further improved, the cost is low, and the method is suitable for large-scale popularization and use.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a nucleic acid-antibody dual cancer detection kit.
Background
Bladder cancer is the most common malignancy in the urinary system and urothelial cancer is the most common type of pathology worldwide. In male malignant tumors, the incidence rate of bladder cancer is 4 th, the mortality rate is 9 th, and the incidence rate ratio of male and female is 3: 1. Over 33 million patients with bladder tumors are newly diagnosed each year, and over 13 million die of bladder cancer. More than 2/3 patients are diagnosed with non-muscle invasive bladder urothelial cancer, but are highly recurrent post-operatively and 10% to 30% of recurrent patients will progress to muscle invasive bladder urothelial cancer, with about 30% of patients diagnosed with distant organ metastasis. Therefore, the method for quickly and accurately detecting the bladder cancer is established, and has great significance for clinical diagnosis and timely and effective treatment of bladder cancer patients.
CXCL1(Chemokine (C-X-C motif) ligand 1), Chemokine CXC motif ligand 1, is a cytokine belonging to the CXC Chemokine family, which was also previously known as GRO1(Human growth-regulated oncogene 1, growth-regulating oncogene 1), KC (Keratinocyte chemoattractant, Keratinocyte chemotaxis), NAP-3 (neutrophiling protein 3, Neutrophil activating protein 3) and MSGA-alpha (Melanoma growth stimulating hormone alpha). In humans, the protein is encoded by the cxcl1 gene and is located on human chromosome 4. CXCL1 is expressed in macrophages, neutrophils and epithelial cells and exhibits neutrophil chemotactic activity, CXCR2 is a chemokine receptor for CXCL and exhibits chemotactic effects upon binding to CXCL 1. Research shows that CXCL1 is highly expressed in bladder cancer, and in the case of poor differentiation, late clinical stage and recurrence, the expression degree is obviously higher than that in the case of good differentiation, early clinical stage and no recurrence, so CXCL1 can be used as a urothelial cell tumor marker for diagnosis, early intervention and treatment of bladder cancer. "W02007026895" discloses the use of CXCL1 protein as a tumor marker for detecting urothelial cancer, and W02010/050554 discloses CXCL1 antibody and a detection kit therefor, but there is still a great need for a new CXCL1 detection kit. Therefore, developing a detection method which is simple to prepare, low in cost, convenient to use and free of a high-precision instrument is an important aspect of the current research.
Disclosure of Invention
Based on the above findings, the primary object of the present invention is to provide a novel bladder cancer detection kit. The invention overcomes the defects of the prior art and provides the detection kit which has the advantages of simple preparation, low cost, convenient use and no need of a high-precision instrument. The kit detects the bladder cancer patient by a nucleic acid-antibody dual detection method, and further improves the detection accuracy.
The invention provides the following technical scheme:
the invention provides a nucleic acid detection reagent aiming at CXCL1, which comprises a detection primer pair and a probe, wherein the sequence of an upstream primer is shown as SEQ ID NO. 2, the sequence of a downstream primer is shown as SEQ ID NO. 3, and the sequence of the probe is shown as SEQ ID NO. 4.
In some embodiments, the probe of the invention is modified with dSpacer at a position 36bp apart from the 5 ' end in the probe, thymine (dT) at positions 34bp and 39bp apart from the 5 ' end on both sides of the dSpacer molecule is replaced with a fluorophore FAM and a quencher BHQ1, respectively, and is modified at the 3 ' end of the probe with a blocking group C3 Spacer.
A monoclonal antibody that specifically binds to CXCL1, comprising a heavy chain variable region comprising a CDR1 region, a CDR2 region and a CDR3 region, the amino acid sequences of the heavy chain CDR1 region, CDR2 region and CDR3 regions are set forth in SEQ ID NOs 7, 8 and 9, respectively; the light chain variable region comprises a CDR1 region, a CDR2 region and a CDR3 region, wherein the amino acid sequences of the light chain CDR1 region, the CDR2 region and the CDR3 region are shown in SEQ ID NOs 10, 11 and 12, respectively.
In another aspect, the invention provides a monoclonal antibody that specifically binds CXCL1, comprising a heavy chain variable region comprising the amino acid sequence SEQ ID No. 5 and a light chain variable region comprising the amino acid sequence SEQ ID No. 6.
In some embodiments, the anti-CXCL 1 antibodies of the invention comprise or consist of two heavy chains and two light chains, wherein each heavy chain comprises a heavy chain constant region sequence, a heavy chain variable region sequence, or a CDR sequence as described above, and each light chain comprises a light chain constant region sequence, a light chain variable region sequence, or a CDR sequence as described above. The antibody of the invention may be a full length antibody comprising a constant region, the full length antibody light chain constant region further comprising murine kappa, lambda chain sequences. The full-length antibody heavy chain constant region further comprises murine IgG1, IgG2a, IgG2b, IgG3, IgA or IgM sequences.
In some embodiments, an anti-CXCL 1 antibody of the invention is a Fab fragment, Fab 'fragment, F (ab')2 fragment, Fv fragment, diabody, linear antibody, single chain antibody molecule, or multispecific antibody formed from an anti-CXCL 1 antibody or antibody fragment described above.
In some embodiments, the anti-CXCL 1 antibody of the invention is used for the preparation of a diagnostic kit for bladder cancer.
In some embodiments, the nucleic acid detection reagent for CXCL1 described in the present invention is used for preparing a bladder cancer diagnostic kit.
In some embodiments, the invention provides a nucleic acid-antibody dual detection kit for bladder cancer, comprising an RPA kit for specifically detecting CXCL1 nucleic acid and an ELISA kit for specifically detecting CXCL1 comprising a monoclonal antibody;
in some embodiments, the invention provides a nucleic acid-antibody dual detection kit for bladder cancer, comprising an RPA kit for specifically detecting CXCL1 nucleic acid and an ELISA kit for specifically detecting CXCL1 comprising a monoclonal antibody; wherein the RPA kit comprises SEQ ID NO:2 and 3 and SEQ ID NO: 4; the ELISA kit comprises an ELISA plate coated by an anti-CXCL 1 monoclonal antibody and an anti-CXCL 1 ELISA-labeled antibody; the anti-CXCL 1 monoclonal antibody includes a heavy chain variable region comprising CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8 and CDR3 shown in SEQ ID NO:9, and a light chain variable region comprising CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11 and CDR3 shown in SEQ ID NO: 12.
In some embodiments, the invention provides a nucleic acid-antibody dual detection kit for bladder cancer, comprising an RPA kit for specifically detecting CXCL1 nucleic acid and an ELISA kit for specifically detecting CXCL1 comprising a monoclonal antibody; wherein the RPA kit comprises SEQ ID NO:2 and 3 and SEQ ID NO: 4; the ELISA kit comprises an ELISA plate coated by an anti-CXCL 1 monoclonal antibody and an anti-CXCL 1 ELISA-labeled antibody; the anti-CXCL 1 monoclonal antibody includes a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO. 5 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO. 6.
Use of a kit for detecting bladder cancer by in vitro determination of the expression of CXCL1 protein, or a gene encoding the protein, in a biological sample from a subject.
In some embodiments, the biological sample of the invention is blood, plasma, serum, urine, bladder tissue or cells.
Advantageous effects
The invention prepares CXCL1 nucleic acid detection reagent by specific RPA primer and probe aiming at CXCL 1; meanwhile, a monoclonal antibody with a good effect is screened and obtained aiming at the CXCL1 antigen, the monoclonal antibody is prepared into a kit for detecting the CXCL1 protein, and the two detection methods are combined for use in the diagnosis of patients with bladder cancer, so that the detection accuracy can be further improved, the cost is low, and the method is suitable for large-scale popularization and use.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.
Figure 1RPA method detects CXCL1 mRNA expression in cells.
Fig. 2CL107 specifically binds to CXCL 1.
Figure 3ELISA method detects CXCL1 protein.
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
Example 1 design of RPA-specific detection primers
The gene sequence of CXCL1 is compared with the homologous sequence thereof, and a more specific region is selected as a target region for primer detection, and the sequence of the region is shown as SEQ ID NO. 1.
According to the rule of RPA primer design and the optimization of the primer, a specific primer sequence is obtained, and the sequence is shown as follows:
upstream primer (SEQ ID NO: 2):
CGAGTGGCACTGCTGCTCCTGCTCCTGGTAGCCG
downstream primer (SEQ ID NO: 3):
CTATGGGGGATGCAGGATTGAGGCAAGCTTTCC
probe sequence (SEQ ID NO: 4):
GAATTCACCCCAAGAACATCCAAAGTGTGAACGTGAAGTCCCCCGGACCC, dSpacer modification is adopted at the position 36bp apart from the 5 ' end in the probe, thymine (dT) at the positions 34bp and 39bp apart from the 5 ' end on both sides of dSpacer molecule is replaced by a fluorescent group FAM and a quenching group BHQ1 respectively, and the 3 ' end of the probe is modified by a blocking group C3 Spacer.
Example 2 detection of CXCL1 Gene expression by RPA method
Separately culturing the bladder cancer cell strain T24 and normal bladder epithelial cell SV-HUC-1. Total RNA from cells was extracted using Trizol reagent (Thermo Co.) and following the instructions. Taking the extracted RNA as a template, carrying out an RPA test, carrying out RPA amplification by using the primer designed in the embodiment 1, and simultaneously setting ultrapure water as a negative control, wherein the reaction time (namely the amplification cycle number) at 40 ℃ is 25min respectively, and the RPA reaction system is 50 mul, wherein 2 mul of forward and reverse primers (10 mul), 2 mul of reverse primer (10 mul), 0.6 mul of probe, 25 mul of buffer solution containing recombinase, DNA polymerase, single-strand binding protein, endonuclease IV and reverse transcriptase, 1 mul of template and 17.9 mul of lddH2O are fully oscillated, uniformly mixed and instantaneously separated, and finally 2.5 mul of 280mM magnesium acetate (MgOAc) is added, and the reaction tube is placed in a real-time fluorescence PCR instrument for a corresponding time at 40 ℃; as shown in fig. 1, the CXCL1 gene was highly expressed in the bladder cancer cell line T24, and CXCL1 expression could be detected in the human normal bladder epithelial cells SV-HUC-1, but the mRNA level of CXCL1 in the bladder cancer cell line was significantly higher than that of the human normal bladder epithelial cells.
Example 3 anti-CXCL 1 antibody preparation
100 μ g of CXCL1 antigen was mixed well with an equal volume of Complete Freund's Adjuvant (CFA) to form a water-like oil bag, and then injected intraperitoneally to initially immunize BALB/C mice. After 3 weeks 100 μ g of CXCL1 was emulsified in equal volumes with Incomplete Freund's Adjuvant (IFA) and boosted by back-injection. Thereafter, the immunization was performed every 2 weeks, and tail blood was collected 7 days after each immunization, and the antibody titer was measured by ELISA. When the antibody titer is appropriate, 100 μ g of CXCL1 antigen and Incomplete Freund's Adjuvant (IFA) are mixed in equal volumes and emulsified, and the last immunization is performed by back multi-point injection. Spleens were harvested on day four and subjected to cell fusion. Spleen cells of immunized Balb/c mice were fused with a myeloma Sp2/0 cell line, and the fused cells were diluted to an appropriate concentration in Iscove's medium (0.1mM hypoxanthine, 0.4. mu.M aminopterin and 16. mu.M thymidine) containing 10% serum, and then cultured in a 96-well plate. After 10 days, cell supernatants were removed and primary cultures showing a positive reaction with CXCL1 protein in the supernatants were examined by high throughput ELISA. The hybridoma cells in the wells were diluted and subcloned, and similarly screened by ELISA, to finally obtain a positive hybridoma cell line, which was named CL-107. After expansion culture, the hybridoma cells were cryopreserved.
EXAMPLE 4 purification of monoclonal antibodies
BALB/c mice were injected intraperitoneally with 0.5 ml/mouse, 1 week before hybridoma inoculation. After 1 week, each mouse was inoculated intraperitoneally at about 1X106(ii) individual hybridoma cells; and after 7-10 days, collecting ascites. Centrifuging ascites at 10000 Xg for 30min, removing precipitate, salting out with 50% ammonium sulfate, coarse extracting, dissolving with PBS, and dialyzing with flowing water for 5 hr; dialyzing and equilibrating with 0.1mol/L phosphate buffer (pH8.0) overnight; and (3) loading, eluting the hybrid protein by using 0.1mol/L phosphate buffer solution (pH8.0), eluting by using citrate eluents with different pH values, collecting elution peaks in sections, and concentrating to obtain the purified anti-CXCL 1 antibody CL-107.
Example 5 anti-CXCL 1 antibody subtype identification
The positive mouse monoclonal cell line selected by indirect ELISA was subjected to subclass measurement using a subclass measuring reagent (Sigma). The microplate provided in the kit had been pre-coated with specific antibodies against mouse IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, kappa light chain, lambda light chain, and the anti-CXCL 1 antibody sample purified in example 4 was added to the sample wells at 50 μ l per well without incubation. Adding 1X goat anti-mouse IgA + IgM + IgG-HRP into sample wells, mixing the sample wells with 50 μ l each, and incubating for 1 h. And (4) deducting liquid in the holes, adding 1XPBST to wash the holes for 3 times, and absorbing the excessive moisture by absorbent paper. Adding color development solution, and developing 100 μ l per well in dark at room temperature for 15 min. The color reaction was stopped by adding 100. mu.l of stop solution. The results showed that the monoclonal antibody of the present invention was a murine monoclonal antibody of the IgG2b type, designated CL-107.
EXAMPLE 6 monoclonal antibody sequencing
The CL-107 hybridoma cell cryopreservation tube is taken out from liquid nitrogen, rapidly thawed at 37 ℃, centrifuged at 1000rpm for 5min to remove the cryopreservation liquid, placed in a 100mm pore plate, cultured until the culture plate accounts for about 80%, added with 1ml Trizol reagent (Thermo company), and extracted with total RNA of hybridoma cells according to the instructions.
Mu.g of the above total RNA was taken, DECP water was added thereto to make the volume 11. mu.l, 1.0. mu.l of oligo (dT) (10. mu.M) was added thereto, 1. mu.l of dNTPs (10mM) was added thereto, the mixture was mixed well, incubated at 65 ℃ for 5 minutes and then placed on ice for 1 minute, followed by addition of 4. mu.l of RT buffer (5X), 1.0. mu.l of DTT (100mM), 1. mu.l of Ribonucleae Inhibitor and 1. mu.l of reverse transcriptase (takara Co., Ltd.), and reacted at 50 ℃ for 10 minutes. The reaction was terminated by incubation at 80 ℃ for 10 minutes, and the obtained cDNA was stored at-20 ℃. Designing specific nested PCR primer, the primer sequence used in the amplification reaction is complementary with the first frame region and the constant region of the antibody variable region, and amplifying the target gene by adopting a conventional PCR method. Wherein the primer sequence is designed according to the literature (Bodo Brocks. Specifes-Cross reactive scFv Against the Tumor promoter Marker "fibrous Activation Protein" Selected by phase Display From an immobilized FAP-/-Knock-Out Mouse).
Sequencing results show that the amino acid sequences of the heavy chain and light chain variable regions of the anti-CXCL 1 antibody CL-107 are respectively shown in SEQ ID NOs: 5 and SEQ ID NO:6 is shown in the specification; the amino acid sequences of 3 CDRs in the heavy chain variable region of the antibody are respectively shown as SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9; the amino acid sequences of 3 CDRs in the light chain variable region are shown as SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12, respectively.
Example 7 identification of antibody specificity
The monoclonal antibody purified in example 4 was cross-reacted with ELISA plates coated with CXCL2, CXCL5 and CXCL8, which are homologous proteins of CXCL1, respectively, by an indirect ELISA method, and the specificity was analyzed. The specific experimental process is that CXCL1, CXCL2, CXCL5 and CXCL8 are respectively coated, the concentration is 0.1mg/ml, the antibodies obtained in the embodiment 3 are respectively added into four different coating antigens, and the degree of cross reaction is detected by indirect ELISA. As shown in fig. 2, the anti-CXCL 1 monoclonal antibody CL-107 reacted only with CXCL1 and did not cross-react with CXCL2, CXCL5 and CXCL 8.
Example 8 anti-CXCL 1 antibody affinity assay
An antibody capture Antibody (AHC) is coated on the surface of a CM5 chip by means of amino coupling, chip activation buffer solution N-ethyl-N' - (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), AHC and ethanolamine for blocking are prepared according to the specifications of an amino coupling kit and an anti-capture kit, a coating program in a Biacore3000 system is selected, and AHC amino is coupled on the surface of a CM5 chip. The anti-CXCL 1 antibody CL-107 obtained in example 4 was captured on the chip surface. The antibody was diluted to 1. mu.g/mL with HBS-EP + buffer, set to a flow rate of 5. mu.l/min, and coated to a response value of 300 RU. CXCL1 antigen was applied in 7 different concentration gradients and serially diluted 2-fold in HBS-EP buffer from 40nM to 0 nM. The CXCL1 antigen flow rate was set at 30. mu.l/min and the binding time was set at 3 min. The flow rate of HBS-EP + buffer was set at 30. mu.l/min, and the dissociation time was set at 10 min. Use of 3M MgCl2As a regeneration buffer, the chip was regenerated according to the regeneration procedure. In addition, affinity assays were performed following the same procedure as for the control antibody IgG1-14 (see CN103408663B patent page 20, line 4). Calculation of binding Rate (K) by Simultaneous fitting of binding and dissociation sensorgramsa) And dissociation Rate (K)d). Equilibrium dissociation constant (K)d) Using dissociation rate (K)d) Rate of binding (K)a) And (4) calculating. The results are shown in table one: the antibody of the invention has high affinity, and the affinity KDValue up to 1.21x10-8M。
Example 9 detection of CXCL1 by ELISA method
Diluting the anti-CXCL 1 monoclonal antibody CL-107 and the control antibody IgG1-14 obtained in example 4 to 2 mu g/ml, and respectively adding the diluted solutions into wells of an enzyme-labeled plate, wherein each well is 100 mu l; the enzyme label plate is placed in an environment of 4 ℃ for coating overnight; adding 100 mul of sealing liquid into each hole, placing in a 37 ℃ incubator for 30 minutes; and taking the enzyme label plate out of the incubator, and then discarding the sealing liquid. The CXCL1 protein was diluted with artificial serum to concentrations of 200, 100, 50, 25, 10, 5, 1pg/ml, respectively. Mu.l of the suspension was added to each well and incubated at 37 ℃ for 1 hour. The supernatant was removed, followed by PBST washing. The CXCL1 antigen is used to immunize rabbit to obtain polyclonal antibody of CXCL1, which is labeled with horseradish peroxidase to obtain enzyme labeled antibody. This was added to wells 100. mu.l per well and incubated at 37 ℃ for 30 minutes in an incubator. Removing supernatant, adding 200 μ l PBST lotion into each well, standing for 2min, removing lotion, and washing for 3 times. Mu.l TMB was added to each well and incubated for 15-30min at room temperature in the dark. The chromogenic reaction was stopped by adding 50. mu.l of stop solution to each well. And (3) placing the ELISA plate into an ELISA reader, and detecting the light absorption value at the wavelength of 450 nm. As shown in FIG. 3, the anti-CXCL 1 monoclonal antibody CL-107 of the present invention has higher signal intensity than the control antibody IgG1-14, and human CXCL1 has higher detection ability.
Sequence listing
<110> Beijing Bao Picture Biotechnology Ltd
<120> nucleic acid-antibody dual cancer detection kit
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 229
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cgagtggcac tgctgctcct gctcctggta gccgctggcc ggcgcgcagc aggagcgtcc 60
gtggccactg aactgcgctg ccagtgcttg cagaccctgc agggaattca ccccaagaac 120
atccaaagtg tgaacgtgaa gtcccccgga ccccactgcg cccaaaccga agtcatagcc 180
acactcaaga atgggcggaa agcttgcctc aatcctgcat cccccatag 229
<210> 2
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cgagtggcac tgctgctcct gctcctggta gccg 34
<210> 3
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ctatggggga tgcaggattg aggcaagctt tcc 33
<210> 4
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gaattcaccc caagaacatc caaagtgtga acgtgaagtc ccccggaccc 50
<210> 5
<211> 119
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Ala
20 25 30
Glu Pro Asp Trp Val Arg Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Asn Ile Ala Gly Pro Thr Asn Lys Glu Tyr Met Thr Asp Ser Val
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Val Tyr
65 70 75 80
Met Glu Phe Arg Ser Leu Thr Leu Glu Asp Ser Ala Ile Tyr Phe Cys
85 90 95
Ala Arg Gly Gly Gln Pro Asp Thr Asn Tyr Met Asn Trp Gly Gln Gly
100 105 110
Thr Ile Phe Thr Val Ser Ser
115
<210> 6
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Asp Val Gln Met Thr Gln Thr Pro Ser Ala Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Ala Arg Ile Thr Pro Lys Gly Asp Thr
20 25 30
Cys Ser Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr Thr Ala Ile Asp Gly Cys Lys Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gly Ala Pro Ser Ile Asn Leu Cys
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 7
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Arg Ala Glu Pro Asp
1 5
<210> 8
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Asn Ile Ala Gly Pro Thr Asn Lys Glu Tyr Met Thr Asp Ser Val Lys
1 5 10 15
Gly
<210> 9
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Gly Gly Gln Pro Asp Thr Asn Tyr Met Asn
1 5 10
<210> 10
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Ala Arg Ile Thr Pro Lys Gly Asp Thr Cys Ser
1 5 10
<210> 11
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Thr Ala Ile Asp Gly Cys Lys
1 5
<210> 12
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Gly Ala Pro Ser Ile Asn Leu Cys Thr
1 5
Claims (9)
1. A nucleic acid-antibody dual bladder cancer detection kit, which comprises an RPA kit for specifically detecting CXCL1 nucleic acid and an ELISA kit containing monoclonal antibody for specifically detecting CXCL 1; wherein the RPA kit comprises SEQ ID NO:2 and 3 and SEQ ID NO: 4; the ELISA kit comprises an ELISA plate coated by an anti-CXCL 1 monoclonal antibody and an anti-CXCL 1 ELISA-labeled antibody; the anti-CXCL 1 monoclonal antibody includes a heavy chain variable region comprising CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8 and CDR3 shown in SEQ ID NO:9, and a light chain variable region comprising CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11 and CDR3 shown in SEQ ID NO: 12.
2. A nucleic acid-antibody dual bladder cancer detection kit, which comprises an RPA kit for specifically detecting CXCL1 nucleic acid and an ELISA kit containing monoclonal antibody for specifically detecting CXCL 1; wherein the RPA kit comprises SEQ ID NO:2 and 3 and SEQ ID NO: 4; the ELISA kit comprises an ELISA plate coated by an anti-CXCL 1 monoclonal antibody and an anti-CXCL 1 ELISA-labeled antibody; the anti-CXCL 1 monoclonal antibody includes a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO. 5 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO. 6.
3. An antibody that specifically binds to CXCL1 antigen, characterized in that the anti-CXCL 1 monoclonal antibody comprises a heavy chain variable region comprising CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8 and CDR3 shown in SEQ ID NO:9, and a light chain variable region comprising CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11 and CDR3 shown in SEQ ID NO: 12.
4. An antibody specifically binding to CXCL1 antigen, characterized in that the anti-CXCL 1 monoclonal antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO. 5 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO. 6.
5. A CXCL1 antigen quantitative detection kit is characterized by comprising an ELISA plate coated by anti-CXCL 1 monoclonal antibody and an anti-CXCL 1 ELISA labeled antibody; the anti-CXCL 1 monoclonal antibody includes a heavy chain variable region comprising CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8 and CDR3 shown in SEQ ID NO:9, and a light chain variable region comprising CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11 and CDR3 shown in SEQ ID NO: 12.
6. A CXCL1 antigen quantitative detection kit is characterized by comprising an ELISA plate coated by anti-CXCL 1 monoclonal antibody and an anti-CXCL 1 ELISA labeled antibody; the anti-CXCL 1 monoclonal antibody includes a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO. 5 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO. 6.
7. The CXCL1 antigen quantitative detection kit according to claim 5 or 6, wherein the anti-CXCL 1 enzyme-labeled antibody is a horseradish peroxidase-labeled anti-CXCL 1 polyclonal antibody.
8. The CXCL1 antigen quantitative detection kit according to any one of claims 5 to 7, wherein the quantitative detection kit further comprises a CXCL1 standard, a wash concentrate, a sample diluent, a substrate solution and a stop solution.
9. Use of the antibody of claim 3 or 4 for the preparation of a bladder cancer detection kit.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110487543.0A CN113186284B (en) | 2021-05-06 | 2021-05-06 | Nucleic acid-antibody dual cancer detection kit |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110487543.0A CN113186284B (en) | 2021-05-06 | 2021-05-06 | Nucleic acid-antibody dual cancer detection kit |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113186284A true CN113186284A (en) | 2021-07-30 |
CN113186284B CN113186284B (en) | 2022-01-07 |
Family
ID=76983474
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110487543.0A Active CN113186284B (en) | 2021-05-06 | 2021-05-06 | Nucleic acid-antibody dual cancer detection kit |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113186284B (en) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103483450A (en) * | 2008-10-31 | 2014-01-01 | 东丽株式会社 | Immunoassay method for human cxcl1 protein |
CN103975078A (en) * | 2011-11-15 | 2014-08-06 | 昂科赛特公司 | Methods and compositions for the treatment and diagnosis of bladder cancer |
-
2021
- 2021-05-06 CN CN202110487543.0A patent/CN113186284B/en active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103483450A (en) * | 2008-10-31 | 2014-01-01 | 东丽株式会社 | Immunoassay method for human cxcl1 protein |
CN103975078A (en) * | 2011-11-15 | 2014-08-06 | 昂科赛特公司 | Methods and compositions for the treatment and diagnosis of bladder cancer |
Non-Patent Citations (2)
Title |
---|
KAWANISHI H等: "Secreted CXCL1 is a potential mediator and marker of the tumor invasion of bladder cancer", 《CLINICAL CANCER RESEARCH》 * |
MAKITO MIYAKE等: "Monoclonal antibody against CXCL1 (HL2401) as a novel Agent in Suppressing IL6 Expression and Tumoral Growth", 《THERANOSTICS》 * |
Also Published As
Publication number | Publication date |
---|---|
CN113186284B (en) | 2022-01-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
HUE027760T2 (en) | Soluble human st-2 antibodies and assays | |
US20230391886A1 (en) | Compositions and methods for muc18 targeting | |
CN114539411B (en) | ROR1 antibody or antigen binding fragment thereof | |
CN112251414A (en) | Hybridoma cell strain, preparation method and application thereof | |
CN113402604A (en) | Kit for rapidly detecting virus and preparation method thereof | |
CN111363037B (en) | Disease detection kit containing antibody specifically binding AFP protein | |
CN102124100A (en) | A monoclonal antibody specifically binding to VEGF and the hybridoma secreting same and uses thereof | |
CN113186284B (en) | Nucleic acid-antibody dual cancer detection kit | |
CN113249445B (en) | Nucleic acid-antibody dual cancer detection kit | |
CN115746131A (en) | Monoclonal antibody for resisting natural rheumatoid factor RF and application thereof | |
CN113896793A (en) | Anti-human IL-17RC monoclonal antibody and application thereof | |
CN112552400B (en) | Leukemia detection kit | |
CN112794913B (en) | Leukemia detection kit | |
CN112710835A (en) | Reproductive system disease detect reagent box | |
CN110579610A (en) | Kit for detecting V-domain immunosuppressive factor activated by T cells | |
CN112876565B (en) | Colorectal cancer detection kit | |
CN112557662B (en) | Colorectal cancer detection kit | |
CN111138541B (en) | Tumor stem cell detection kit | |
CN112500488B (en) | Cancer detection kit | |
CN117683121B (en) | Anti-varicella-zoster virus antibodies and uses thereof | |
CN113278075B (en) | Inflammation detection kit | |
CN113402605B (en) | Rapid inflammation detection kit | |
CN113846066B (en) | Hybridoma cell strain secreting ST2 antibody and application thereof | |
CN116284411B (en) | Antibody against recombinant human coagulation factor VIII-Fc fusion protein and application thereof | |
CN112684175B (en) | Kit for detecting ovarian cancer |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
TA01 | Transfer of patent application right | ||
TA01 | Transfer of patent application right |
Effective date of registration: 20211220 Address after: 313300 No. 3787 eastern section of sunshine Avenue, Anji County, Huzhou, Zhejiang. Applicant after: ZHEJIANG ORIENT GENE BIOTECH CO.,LTD. Address before: 804, 8 / F, building 23, yard 10, Rongcheng North Road, Huairou District, Beijing Applicant before: Beijing Baotu Biotechnology Co.,Ltd. |
|
GR01 | Patent grant | ||
GR01 | Patent grant |