CN111234023B - Small cell lung cancer detection kit - Google Patents
Small cell lung cancer detection kit Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- 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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- 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
- G01N33/57423—Specifically defined cancers of lung
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- 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
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- 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
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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- 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
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- 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]
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- 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
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/988—Lyases (4.), e.g. aldolases, heparinase, enolases, fumarase
Abstract
The invention relates to a small cell lung cancer detection kit, which comprises an antibody capable of being specifically combined with NSE, has higher sensitivity, and is used for early screening diagnosis, curative effect detection and prognosis judgment of small cell lung cancer.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a small cell lung cancer detection kit.
Background
Lung cancer is one of the most common malignant tumors, and is also the most lethal tumor. 2018 global cancer statistics, there are about 1810 new cancer cases and 960 ten thousand cancer patient deaths, with lung cancer accounting for 11.6% of all new cancer cases and 18.4% of all cancer total deaths. Pathologically, lung cancer can be classified into Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC). The Small Cell Lung Cancer (SCLC) is a neuroendocrine tumor, accounts for 15% -20% of all lung malignant tumors, has the characteristics of high malignancy, strong invasiveness, easy relapse and metastasis, limits the long-term survival of patients, and is a malignant tumor with extremely poor prognosis. In clinic, most lung cancer is diagnosed at the middle and late stage, the optimal operation time is missed, and the 5-year survival rate is less than 20%, so the early diagnosis has very important significance for controlling the lung cancer. With the research progress of molecular biology, immunology and pathogenesis of lung cancer, the detection of serum tumor markers has been widely applied to early screening diagnosis, curative effect detection and prognosis judgment of lung cancer due to the advantages of simple operation, good repeatability and small wound. Currently, the most commonly used tumor markers for lung cancer serum in clinic include nerve-specific enolase (NSE), carcinoembryonic antigen (CEA), cytokeratin 19 fragment (CYFRA 211), carbohydrate antigen 125 (CA 125), cancer-associated carbohydrate antigen (CA 199), and the like.
Neuron-specific enolase (NSE) is an isozyme of enolase. Enolase isozymes can be classified into five dimer isozymes of α α α, β β, γ γ, α β and α γ according to the differences of three subunits of α, β and γ. The alpha subunit is mainly present in tissues such as liver, kidney and the like, and is called enolase of a non-nervous system (NNE); the β subunit is found predominantly in skeletal and cardiac muscle and is referred to as muscle-specific enolase (MSE); the gamma subunit is present predominantly in neural tissue. The isozyme composed of gamma and alpha gamma is specific to neurons and neuroendocrine cells, so the isozyme is named as neuron-specific enolase. NSE is widely distributed in human and various animals in mature neurons, neuroendocrine cells of peripheral nerves and some sensory cells, and is present in the highest amount in normal human brain tissue, and tumor tissue derived from neuroendocrine cells is also abnormally expressed. It is commonly found in neuroblastoma, small cell lung cancer, pancreatic cancer, etc. Thus NSE becomes a tumor marker for Small Cell Lung Cancer (SCLC) in the typing of lung cancer.
Several patents also disclose NSE antibodies, such as: CN105132380A, CN103087193A, and CN102435748A, but the inventors found that these antibodies still have to be improved in sensitivity after the study.
Disclosure of Invention
Based on the above findings, the primary object of the present invention is to provide a novel NSE antibody with higher sensitivity, so as to solve the problem of low sensitivity of the existing NSE antibody.
The invention provides the following technical scheme:
an anti-NSE antibody, consisting of a heavy chain and a light chain comprising a variable region and a constant region, respectively, wherein the heavy chain variable region is selected from SEQ ID NO 13 and 14 and the light chain variable region is selected from SEQ ID NO 15 and 16.
The heavy and light chain variable region of the antibody of the present invention has 6 complementarity determining regions. 3 CDR sequences of the heavy chain variable region of the NSE antibody are respectively SEQ ID NO 1, SEQ ID NO 3 and SEQ ID NO 5; the variable region of light chain has 3 CDR sequences of SEQ ID NO 7, SEQ ID NO 9 and SEQ ID NO 11.
3 CDR sequences of the heavy chain variable region of the NSE antibody are respectively SEQ ID NO. 2, SEQ ID NO. 4 and SEQ ID NO. 6; the variable region of light chain has 3 CDR sequences of SEQ ID NO 8, 10 and 12.
The heavy and light chains of the NSE antibodies of the invention further comprise constant regions, and the antibody light chain constant regions further comprise murine kappa and lambda chain sequences. The antibody heavy chain constant region further comprises murine IgG1, IgG2a, IgG2b, or IgG3, or IgM sequences.
The invention aims to provide two novel NSE antibodies, wherein the antibodies specifically recognize different epitopes of NSE and are used for detecting the presence and the content of NSE protein in a sample.
It is another object of the present invention to provide a method for detecting NSE using two or more different NSE antibodies that specifically recognize different epitopes of NSE, said epitopes being located at 89-97aa and 285-298aa, respectively, of the NSE protein. The method is a double antibody sandwich Elisa assay.
Still another objective of the present invention is to provide a more sensitive NSE antibody kit, which contains the above two antibodies recognizing different NSE epitopes, and is used for early screening diagnosis, therapeutic effect detection and prognosis judgment of small cell lung cancer.
The invention has the following beneficial effects: the strategy of preparing monoclonal antibody by using linear epitope and carrier protein as antigen can conveniently and quickly obtain new NSE monoclonal antibody for identifying different epitopes; the NSE detection kit established by the antibody has higher sensitivity, and provides help for the early diagnosis of the small cell lung cancer.
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.
FIG. 1 shows the subtype identification of monoclonal antibodies.
FIG. 2 shows that Western-blotting was used to detect the expression of NSE in A549 cells and Hela cells for two antibodies.
FIG. 3 is a graph of the sensitivity detection of the human NSE double antibody sandwich Elisa kit.
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 preparation of design, Synthesis and conjugation of NSE Polypeptides
The linear epitope of NSE is predicted by using three-dimensional structure prediction software of protein, and finally KLDNLMLEL (89-97 aa) RDYPVVSIEDPFDQ (285-298 aa) 2 polypeptide sequences are selected as antigen sequences. To facilitate coupling to the carrier protein, the synthetic polypeptide has a cysteine added at the C-terminus, i.e.: KLDNLMLELC, RDYPVVSIEDPFDQC are provided. The polypeptide was synthesized by Nanjing Kinsrui Biotech. The chemically synthesized polypeptide antigen is a small molecule, is difficult to have good antigenicity and can only induce weak immune response of animals such as cows, so that the immunogenicity of the polypeptide needs to be further enhanced by a method of coupling carrier protein. The carrier protein contains many epitopes and is capable of stimulating T helper cells and thereby inducing B cell responses. There are many carrier proteins for cross-linking with polypeptides, the most commonly used carriers being Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA), Ovalbumin (OVA). KLH has higher antigenicity, so the KLH is selected as a polypeptide cross-linking carrier. Adopting bifunctional coupling agent glutaraldehyde for coupling, dissolving the NSE polypeptide protein and KLH (purchased from Sigma) in PBS, uniformly mixing in a coupling bottle, slowly dropwise adding 0.25% glutaraldehyde solution into the mixed solution, reacting for 4h at 4 ℃, placing the reaction solution in PBS for dialysis at 4 ℃, finally obtaining chemically coupled NSE-KLH, naming the antigen containing KLDNLMLEL polypeptide as NSE-E1, and naming the antigen containing RDYPVVSIEDPFDQ polypeptide as NSE-E2.
Example 2 anti-NSE antibody preparation and purification
At the time of primary immunization, the administration will be carried outThe antigens NSE-E1 and NSE-E2 (the concentration is 1 mug/mul) prepared in example 1 are respectively and fully mixed with equal volume of Complete Freund's Adjuvant (CFA) to form a water-in-oil state, and the water-in-oil state is injected into a BALB/C mouse at 200 mul/abdominal cavity. And performing first strengthening after 3 weeks, mixing and emulsifying antigens NSE-E1 and NSE-E2 (the concentration is 1 mug/mug) respectively with an Incomplete Freund's Adjuvant (IFA) in an equal volume, and injecting the mixture at multiple points on the back of 200 mug/mouse. The subsequent booster immunization was performed every 2 weeks, and tail blood was collected 7 days after each immunization to detect the antibody titer by ELISA. When the antibody titer reaches 10-4On the left and right, NSE-E1 and NSE-E2 antigens (the concentration is 1 mu g/mu l) and Incomplete Freund's Adjuvant (IFA) are mixed uniformly in equal volume and emulsified, and the back of 200 mu l/mouse is injected at multiple points to strengthen the immunity once. Spleens were harvested on day four and subjected to cell fusion. Taking splenocytes of immunized Balb/c mice, fusing the splenocytes with myeloma Sp2/0 cell strain, adding HAT into fused cells for plating, changing the liquid for half after 3 days, changing the liquid for one week, and changing HAT culture medium for culture. After 12 days, cell supernatants were removed and primary cultures showing positive reaction with NSE protein in the supernatants were assayed by high throughput ELISA. And then, the hybridoma cells in the hole are diluted and subcloned, and are screened by an ELISA method, finally, a positive hybridoma cell strain E1-7E2 is obtained from an NSE-E1 antigen group, and a positive hybridoma cell strain E2-4F6 is obtained from an NSE-E2 antigen group. Respectively culturing hybridoma cell lines E1-7E2 and E2-4F6 in an enlarged manner, collecting the hybridoma cells, resuspending with PBS, and configuring into 107Hybridoma cell suspension in ml. Freund's incomplete adjuvant was used as an inducer. Injecting Freund's incomplete adjuvant into Balb/c abdominal cavity for about 8 weeks, and injecting 500ul of 10-concentration adjuvant into Balb/c mouse abdominal cavity after 3 days7Hybridoma cell suspension in ml. When the abdomen of the mouse swells, ascites purified antibodies are collected. And (3) precipitating by using saturated ammonium sulfate (PH = 7.8) to obtain crude NSE monoclonal antibody, and performing protein G column chromatography to obtain NSE antibodies E1-7E2 and E2-4F6 with higher purity.
Example 3 anti-NSE antibody subtype identification
The subtype of the anti-NSE monoclonal antibody obtained in example 2 was identified by ELISA (kit purchased from Proteitech). Sample adding: the microplate provided in the kit was already pre-coated with specific antibodies against mouse IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, kappa light chain, lambda light chain, and the anti-NSE antibody samples E1-7E2 and E2-4F6 purified in example 2 were added to sample wells, 50. mu.l per well, respectively, without incubation. Incubation of enzyme-labeled antibody: 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. Washing: and (4) deducting liquid in the holes, adding 1XPBST to wash the holes for 3 times, and absorbing the excessive moisture by absorbent paper. Color development: adding color development solution, and developing 100 μ l per well in dark at room temperature for 15 min. And (4) terminating: the color reaction was stopped by adding 100. mu.l of stop solution. Reading value: the OD value at 450nm is detected by a microplate reader, and the result is shown in figure 1, the heavy chain subtypes of the anti-NSE monoclonal antibodies E1-7E2 and E2-4F6 are IgG1, and the light chain thereof is Kappa.
Example 4 Western-blotting detection of antibody binding to NSE protein
The anti-NSE antibodies E1-7E2 and E2-4F6 prepared in example 2 were detected and analyzed by Western-blotting (WB) method. After the recovery, the human lung cancer cell A549 and the cervical cancer cell Hela are subjected to subculture, wherein the A549 cell expresses NSE and serves as a positive experimental group, and the Hela cell does not express NSE and serves as a negative control group. Sample preparation: a549 and Hela cells are cultured for 24h, then digested and harvested, and centrifuged at 1000rpm for 3 minutes. PBS was added for washing once, and the supernatant was discarded by centrifugation. The cell pellet was resuspended in 100ul PBS, and an equal amount of 4XSDS lysate was added and mixed well. Boiling the sample in water for 10min, centrifuging at 12000rpm, and then obtaining a WB sample after 10 min. SDS-PAGE electrophoresis: preparing 10% separation gel. Pouring Tris-glycine electrophoresis buffer solution into the electrophoresis tank, and sampling according to 20 mu l/hole. During electrophoresis, the voltage applied to the concentrated gel is 80V
When the leading edge of the dye enters the separation gel, the voltage is changed to 120V. Semi-dry transfer membrane method: the filter paper, the cellulose membrane, the gel and the filter paper are overlapped in sequence, and then air bubbles are removed. The rotary die instrument is opened, the voltage is set to be 18V, and the time is 1 h. Proteins in WB samples were transferred to PVDF membranes. And (3) sealing: incubate 5% skim milk for 1h at room temperature and seal. Incubating the primary antibody: anti-NSE antibodies E1-7E2, E2-4F6 were incubated overnight as primary antibodies, respectively. Washing: adding TBST membrane washing solution, washing for 3 times, placing on 40rpm shaking table each time, and washing for 5 min. Incubation of secondary antibody: HRP conjugated goat anti-mouse secondary antibody was added for incubation and incubated for 1h at room temperature. Washing: the antibody bound non-specifically was washed off 3 times in the same manner. And (3) developing: adding color developing solution, and imaging in a developing instrument. As shown in FIG. 2, the anti-NSE antibodies E1-7E2 and E2-4F6 both detected the NSE protein in A549 cells, but not in Hela cells. The anti-NSE antibodies E1-7E2, E2-4F6 were shown to be capable of specifically binding to NSE proteins.
Example 5 detection of double antibody Sandwich ELISA kit sensitivity Using NSE
Using the NSE antibody prepared in example 2, a double antibody sandwich ELISA kit was prepared, and the sensitivity of the double antibody sandwich ELISA kit was detected using NSE. Antibody coating: the anti-NSE antibody E1-7E2 was used as a coating antibody, diluted to 0.5. mu.g/ml with a coating solution, 100. mu.l/well, incubated overnight at 4 ℃ and immobilized on an ELISA plate. Washing: removing supernatant, adding 200 μ l PBST lotion into each well, standing for 2min, removing lotion, and washing repeatedly for 3 times, wherein the elisa plate is dried as much as possible. And (3) sealing: 2% casein-water was used as blocking solution, 100. mu.l was added to each well, and blocking was performed at room temperature for 1 hour. Washing: removing supernatant, adding 200 μ l PBST lotion into each well, standing for 2min, removing lotion, and washing repeatedly for 3 times, wherein the elisa plate is dried as much as possible. Incubation of antigen: NSE antigen (purchased from Beijing Boaosen Biotechnology Co., Ltd.) was diluted in a gradient of 800ng/ml, 200ng/ml, 50ng/ml, 12.5ng/ml, 3.125ng/ml,0.781 ng/ml, 0.195 ng/ml,0.0488 ng/ml. The diluted antigen was added to the ELISA plate in sequence, 100. mu.l per well, and incubated at 37 ℃ for 30 min. Washing: removing supernatant, adding 200 μ l PBST lotion into each well, standing for 2min, removing lotion, and washing repeatedly for 3 times, wherein the elisa plate is dried as much as possible. Incubation of enzyme-labeled antibody: horseradish peroxidase (HRP) -labeled anti-NSE antibody E2-4F6, i.e., E2-4F6-HRP, was used as a detection antibody, and the antibody was diluted to 0.5 μ g/ml, 100 μ l was added to each well, and incubated at room temperature for 1 h. Washing: removing supernatant, adding 200 μ l PBST lotion into each well, standing for 2min, removing lotion, and washing repeatedly for 3 times, wherein the elisa plate is dried as much as possible. Color development: mu.l TMB was added to each well and incubated for 15-30min at room temperature in the dark. And (4) terminating: the chromogenic reaction was stopped by adding 50. mu.l of stop solution to each well. Reading value: and (3) placing the ELISA plate into an ELISA reader, and detecting the light absorption value at the wavelength of 450 nm. The resulting data are shown in FIG. 3, with a detectable range of 48.8pg/ml to 50 ng/ml. According to the prior art, for example, as shown by the sensitivity result in CN105132380A, the lowest concentration detected by the method is 240 pg/ml. Therefore, the sensitivity of the double-sandwich ELISA detection kit prepared by the NSE antibody is obviously higher than that of the kit in the prior art.
EXAMPLE 6 monoclonal antibody sequencing
Cell preparation: hybridoma cell lines E1-7E2, E2-4F6 were cultured to a total amount of 107The cells were collected by centrifugation at 1000rpm for 5 min. RNA extraction: TRNzol-A + was added to the cell pellet for lysis and allowed to stand at room temperature for 15 min. Mu.l chloroform was added to each ml of TRNzol-A +, vortexed for 15 seconds, and allowed to stand for 3 minutes. 13000 rpm, 4 ℃ for 10 minutes, Trizol-A + cell solution is divided into three layers: transferring the water phase dissolved with the RNA into a centrifuge tube, adding isopropanol with the same volume into the water phase, uniformly mixing, and standing at room temperature for 25 minutes. 13000 rpm, 4 ℃ for 10 minutes, discard the waste liquid to get the bottom RNA precipitation. After washing the RNA pellet twice with 75% ethanol, the RNA was dissolved in PEDC water and stored at-80 ℃. Reverse transcription: synthesizing first chain cDNA by reverse transcription kit SMARTERRACE, and amplifying antibody variable region DNA sequence corresponding to hybridoma cell by using the first chain cDNA as subsequent template. Amplification: according to the subtype identification result of example 3, IgG1 and IgG2b subtype heavy chain and Kappa light chain constant region sequences are obtained, specific nested PCR primers are designed, the primer sequences used in the amplification reaction are complementary with the first framework region and the constant region of the antibody variable region, and the target gene is amplified by adopting a conventional PCR method. Sequencing: after sequencing the amplified products, the heavy chain variable region sequence of the anti-NSE antibody E1-7E2 secreted by hybridoma clone E1-7E2 is SEQ ID NO: 13 and light chain variable region sequences SEQ ID NO: 15; the 3 CDR sequences of the heavy chain variable region of the antibody are respectively SEQ ID NO. 1, SEQ ID NO. 3 and SEQ ID NO. 5; the variable region of light chain has 3 CDR sequences of SEQ ID NO 7, SEQ ID NO 9 and SEQ ID NO 11. Secreted by hybridoma clone E2-4F6The heavy chain variable region sequence of anti-NSE antibody E2-4F6 SEQ ID NO: 14 and light chain variable region sequence SEQ ID NO: 16; the 3 CDR sequences of the heavy chain variable region of the antibody are respectively SEQ ID NO. 2, SEQ ID NO. 4 and SEQ ID NO. 6; the variable region of light chain has 3 CDR sequences of SEQ ID NO 8, 10 and 12.
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Claims (7)
1. A small cell lung cancer detection kit is characterized in that: comprising an antibody that specifically binds to NSE, said antibody being any one of:
a) the 3 CDR sequences of the heavy chain variable region of the antibody are respectively SEQ ID NO 1, SEQ ID NO 3 and SEQ ID NO 5; the variable region of the light chain has 3 CDR sequences of SEQ ID NO 7, SEQ ID NO 9 and SEQ ID NO 11;
b) the 3 CDR sequences of the heavy chain variable region of the antibody are respectively SEQ ID NO 2, SEQ ID NO 4 and SEQ ID NO 6; the variable region of light chain has 3 CDR sequences of SEQ ID NO 8, 10 and 12.
2. A small cell lung cancer detection kit is characterized in that: comprising an antibody that specifically binds to NSE, said antibody being any one of:
a) the heavy chain variable region sequence of the antibody is shown as SEQ ID NO. 13, and the light chain variable region sequence is shown as SEQ ID NO. 15;
b) the heavy chain variable region sequence of the antibody is shown as SEQ ID NO. 14, and the light chain variable region sequence is shown as SEQ ID NO. 16.
3. A small cell lung cancer detection kit is characterized in that: the kit is a double-antibody sandwich ELISA kit, and comprises two different antibodies specifically bound with NSE, wherein the sequences of the two different antibodies are respectively as follows:
a) the 3 CDR sequences of the heavy chain variable region of the antibody are respectively SEQ ID NO 1, SEQ ID NO 3 and SEQ ID NO 5; the variable region of the light chain has 3 CDR sequences of SEQ ID NO 7, SEQ ID NO 9 and SEQ ID NO 11;
b) the 3 CDR sequences of the heavy chain variable region of the antibody are respectively SEQ ID NO 2, SEQ ID NO 4 and SEQ ID NO 6; the variable region of light chain has 3 CDR sequences of SEQ ID NO 8, 10 and 12.
4. A small cell lung cancer detection kit is characterized in that: the kit is a double-antibody sandwich ELISA kit, and comprises two different antibodies specifically bound with NSE, wherein the sequences of the two different antibodies are respectively as follows:
a) the heavy chain variable region sequence of the antibody is shown as SEQ ID NO. 13, and the light chain variable region sequence is shown as SEQ ID NO. 15;
b) the heavy chain variable region sequence of the antibody is shown as SEQ ID NO. 14, and the light chain variable region sequence is shown as SEQ ID NO. 16.
5. An antibody that specifically binds NSE, characterized by: it is any one of the following:
a) the 3 CDR sequences of the heavy chain variable region of the antibody are respectively SEQ ID NO 1, SEQ ID NO 3 and SEQ ID NO 5; the variable region of the light chain has 3 CDR sequences of SEQ ID NO 7, SEQ ID NO 9 and SEQ ID NO 11;
b) the 3 CDR sequences of the heavy chain variable region of the antibody are respectively SEQ ID NO 2, SEQ ID NO 4 and SEQ ID NO 6; the variable region of the light chain has 3 CDR sequences of SEQ ID NO 8, SEQ ID NO 10 and SEQ ID NO 12;
c) the heavy chain variable region sequence of the antibody is shown as SEQ ID NO. 13, and the light chain variable region sequence is shown as SEQ ID NO. 15;
d) the heavy chain variable region sequence of the antibody is shown as SEQ ID NO. 14, and the light chain variable region sequence is shown as SEQ ID NO. 16.
6. Use of an antibody according to claim 5 for the preparation of a kit for the detection of NSE.
7. Use of the antibody of claim 5 for the preparation of a kit for the detection of small cell lung cancer.
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| CN112646889B (en) * | 2020-12-29 | 2021-08-20 | 上海奕检医学检验实验室有限公司 | Rapid lung cancer detection kit combining rapid nucleic acid screening and antibody detection |
| CN113087802B (en) * | 2021-04-07 | 2022-10-11 | 浙江格蕾斯生物科技有限公司 | Kit for detecting cancer by combining nucleic acid detection with antibody |
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