CN118005793A - Monoclonal antibody specifically binding to human CD14 and application thereof - Google Patents
Monoclonal antibody specifically binding to human CD14 and application thereof Download PDFInfo
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- 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
- G01N33/57488—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 involving compounds identifable in body fluids
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- C07K2317/00—Immunoglobulins specific features
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Abstract
The invention discloses a monoclonal antibody specifically binding to human CD14, which comprises two antibodies, namely sCD14/2D11 and sCD14/3G3, wherein the sCD14/2D11 and the sCD14/3G3 can both recognize and bind to polypeptide chains of the amino acid sequence of CD 14. Or comprises two antibodies, namely sCD14/2D11 and HRP-sCD14/3G3, wherein the HRP-sCD14/3G3 is HRP-labeled sCD14/3G3. Meanwhile, the invention also discloses application of the monoclonal antibody specifically combined with human CD14 in preparing diagnostic reagents for detecting human soluble CD 14. The monoclonal antibody provided by the invention can identify and combine the polypeptide chain of the amino acid sequence of CD14, can be used for detecting human CD14, and has important significance for detecting and diagnosing infectious diseases and liver cancer.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a monoclonal antibody specifically binding to human CD14 and application thereof.
Background
CD14 is a Lipopolysaccharide (LPS) receptor, a leukocyte differentiation antigen that exists with the surface of monocytes, macrophages and dendritic cells, and was first discovered on the surface of any monocytes in 1981. The human CD14 encoding gene is located in the 5q23-q31 region of the long arm end of chromosome 5 and encodes a 375 amino acid polypeptide chain. Wright et al in 1990 elucidated that CD14 acts as an LPS receptor, mediating the LPS response in the organism.
CD14 molecules include two molecular types, membrane-bound CD14 (mCD 14) and soluble CD14 (sCD 14), mCD14 is a glycoprotein, which is predominantly distributed on the cell surface of monocytes, macrophages, anchored to the cell membrane by glycosyl phosphatidylinositol. The sCD14 protein structure is substantially the same as that of mCD14, but does not contain phosphatidylinositol structure, and is found in human plasma for the first time in 1985 by researchers such as MALISZEWSKI. sCD14 in humans can be produced by two pathways: first, the decomposition reaction of mCD14 is catalyzed by the protease or phosphatidylkinase of the body, and the phosphatidylinositol structure is removed, so that mCD14 falls off from the cell membrane to the blood to form sCD14; second, CD14 gene is transcribed and translated into CD14 protein, but is directly secreted into the blood without phosphatidylinositol modification to form sCD14.
CD14 can bind to LPS, mediating monocyte and macrophage immune responses. Studies have shown that sCD14 levels are elevated in serum from a variety of patients with sepsis, rheumatoid arthritis, liver disease, systemic lupus erythematosus, allergic dermatitis, and the like. Sylvette Bas et al report that sCD14 can act as an Acute Phase Protein (act-Phase Protein) that, together with C-reactive Protein (CRP), reacts with the inflammatory state of the body. However, no method for detecting the concentration of sCD14 in serum is applied to clinical diagnosis of related diseases at present.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a monoclonal antibody specifically binding to human CD14 and application thereof, wherein the monoclonal antibody specifically binding to human CD14 can be used for preparing diagnostic reagents for infectious diseases and liver cancer.
A monoclonal antibody that specifically binds human CD14, comprising two antibodies, sCD14/2D11 and sCD14/3G3, respectively, each of said sCD14/2D11 and sCD14/3G3 being capable of recognizing and binding to a polypeptide chain of the amino acid sequence of CD 14;
The amino acid sequence of the light chain variable region of sCD14/2D11 is shown as SEQ ID NO. 2, and the amino acid sequence of the heavy chain variable region of sCD14/2D11 is shown as SEQ ID NO. 3;
The amino acid sequence of the light chain variable region of sCD14/3G3 is shown as SEQ ID NO. 4, and the amino acid sequence of the heavy chain variable region of sCD14/3G3 is shown as SEQ ID NO. 5.
Preferably, the amino acid sequence of the CD14 is shown as SEQ ID NO. 1.
A monoclonal antibody that specifically binds to human CD14, comprising two antibodies, sCD14/2D11 and HRP-sCD14/3G3, respectively, each of said sCD14/2D11 and HRP-sCD14/3G3 being capable of recognizing and binding to a polypeptide chain of the amino acid sequence of CD 14;
The HRP-sCD14/3G3 is HRP-marked sCD14/3G3;
The amino acid sequence of the light chain variable region of sCD14/2D11 is shown as SEQ ID NO. 2, and the amino acid sequence of the heavy chain variable region of sCD14/2D11 is shown as SEQ ID NO. 3;
The amino acid sequence of the light chain variable region of sCD14/3G3 is shown as SEQ ID NO. 4, and the amino acid sequence of the heavy chain variable region of sCD14/3G3 is shown as SEQ ID NO. 5.
Preferably, the amino acid sequence of the CD14 is shown as SEQ ID NO. 1.
The monoclonal antibody specifically binding to human CD14 is applied to the preparation of diagnostic reagents for detecting human soluble CD 14.
Preferably, the diagnostic reagent for detecting human soluble CD14 is a diagnostic reagent for detecting soluble CD14 in human serum or plasma.
Preferably, the diagnostic reagent for detecting human soluble CD14 is used for preparing a detection reagent for infectious diseases or a detection reagent for liver cancer.
Preferably, the infectious disease is a bacterial infection.
The invention has the advantages that:
The monoclonal antibody provided by the invention can identify and combine the polypeptide chain of the amino acid sequence of CD14, can be used for detecting human CD14, and has important significance for detecting and diagnosing infectious diseases and liver cancer.
Drawings
FIG. 1 shows the purification results of sCD14/2D11 monoclonal antibody by SDS-PAGE.
FIG. 2 shows the purification results of sCD14/3G3 monoclonal antibody by SDS-PAGE.
FIG. 3 shows the result of detection of sCD14/2D11 monoclonal antibody and human CD14 by using a western-blot method.
FIG. 4 shows the result of detecting the reaction of sCD14/3G3 monoclonal antibody with human CD14 by using a western-bolt method.
FIG. 5A standard curve was obtained by detecting human CD14 standard using sCD14/2D11 and HRP-sCD 14/3G3 double antibody sandwich method.
FIG. 6 results of detection of soluble CD14 in serum of infected patients and healthy persons using sCD14/2D11 and HRP-sCD 14/3G3 double antibody sandwich.
FIG. 7 shows the results of detection of soluble CD14 in serum of liver cancer and healthy persons using sCD14/2D11 and HRP-sCD 14/3G3 double antibody sandwich method.
FIG. 8 uses a subject work (ROC) curve analysis to test the diagnostic efficacy of human serum soluble CD14 for infected patients in accordance with the methods of the present invention.
FIG. 9 uses a subject work (ROC) curve analysis to test the diagnostic efficacy of human serum soluble CD14 in liver cancer patients according to the method of the present invention.
Detailed Description
The project of the invention is funded by talent funds in Tang-Hospital (2021 SHRC 004).
The invention adopts human CD14 to immunize BALB/c mice, prepares a group of hybridoma cell strains secreting mouse anti-human CD14 monoclonal antibodies, screens out hybridoma cell strains which can stably secrete high-affinity monoclonal antibodies sCD14/2D11 and sCD14/3G3, prepares ascites and purifies monoclonal antibodies; extracting RNA of the hybridoma cell strain, reversely transcribing the RNA into cDNA, and carrying out PCR amplification to obtain a light chain variable region sequence and a heavy chain variable region sequence of the monoclonal antibody, and comparing to confirm the uniqueness of the sequences; labeling sCD14/3G3 monoclonal antibody by using HRP; the human serum soluble CD14 is detected by using sCD14/2D11 and HRP-sCD14/3G3 double antibody sandwich method, and the specific steps are as follows:
1. preparation and identification of sCD14/2D11 and sCD14/3G3 monoclonal antibodies
1.1 Immunization of animals
8-Week-old BALB/c mice (purchased from the university of medical laboratory animal center of air force army) were immunized with human CD14 protein (amino acid sequence shown in SEQ. ID. NO.1, purchased from Yinqiao Shenzhou) for primary immunization: 50 μg CD14 was mixed with 150 μl Freund's complete adjuvant in equal volumes and injected subcutaneously in the back at multiple points; four weeks later, secondary immunization was performed: mouse serum antibody titers were determined after 20 days by subcutaneous multipoint injection on the back using 50 μg CD14 in equal volume with 150 μl of incomplete freund's adjuvant; after 1 week, 50 μg CD14 was injected intraperitoneally to boost, and after 3 days mice were sacrificed and spleens were removed in preparation for cell fusion.
1.2 Hybridoma cell preparation
The immune mouse spleen cell suspension is fused with mouse myeloma cell SP2/0 by using PEG1500 as a fusion agent. The fused cells were inoculated into a 96-well cell culture plate containing feeder cells (6-week-old BALB/c murine thymocytes) and cultured in 1640 medium containing 1% HAT and 20% FBS. When the clone grows to 1/3-1/2 bottom area, collecting culture supernatant, detecting antibody in the culture supernatant by an Elisa method, screening hybridoma clones secreting anti-CD 14 monoclonal antibody, and obtaining 2 hybridoma cell strains capable of stably secreting monoclonal monomers, wherein the hybridoma cell strains are respectively marked as 2D11 and 3G3.
1.3SCD14/2D11 and sCD14/3G3 monoclonal antibody purification
2D11 and 3G3 hybridoma cells were cultured, and ascites was prepared by injecting 1X 10 6 cells into the abdominal cavity of a BALB/c mouse. Monoclonal antibodies in ascites were purified by an n-octanoic acid-ammonium sulfate precipitation method, and antibodies secreted by 2D11 and 3G3 were designated sCD14/2D11 and sCD14/3G3, respectively, and the purified antibodies were detected by SDS-PAGE, and the purification method was as follows: extracting ascites from mice, centrifuging at 3000r/min for 5min, removing blood cells, centrifuging the supernatant at 15000r/min for 30min, filtering the supernatant at 0.22 μm, and slowly adding acetate buffer solution with volume of 3 times; then, n-octanoic acid (25. Mu.L of ascites 1 mL) was slowly added to the above solution, stirred at room temperature for 10min, centrifuged at 4℃and 10000rpm for 20min, the precipitate was discarded, and the volume of the supernatant was measured; 1/10 volume of 10 XPBS buffer was added to the supernatant, the pH was adjusted to 7.4, and the ratio was 1:1 proportion of saturated ammonium sulfate solution is slowly added into the mixture, and the mixture is stood and precipitated overnight at 4 ℃; the next day, the whole liquid was poured into a centrifuge tube, and after centrifugation, the pellet was resuspended in PBS, and the results are shown in FIGS. 1 and 2;
as can be seen from FIGS. 1 and 2, the sCD14/2D11 and sCD14/3G3 monoclonal antibodies with higher purity are obtained by precipitation and purification of n-octanoic acid-ammonium sulfate.
1.4 Identification of sCD14/2D11 and sCD14/3G3 monoclonal antibodies
Human CD14 protein is taken as antigen, and the binding between sCD14/2D11 and sCD14/3G3 monoclonal antibodies and CD14 in cells is detected by a western-blot method, which comprises the following steps: the sample is loaded after the CD14 protein is boiled, 26mA is used for transferring films for 2.5 hours, and 5% skimmed milk is used for sealing for 2 hours; after TBST film washing, the method comprises the following steps of 1: adding sCD14/2D11 and sCD14/3G3 monoclonal antibodies serving as primary antibodies in a proportion of 1000, and incubating overnight at 4 ℃; the following day is 1:2000 proportion of goat anti-mouse IgG-HRP antibody is added as secondary antibody, the secondary antibody is incubated for 1h at room temperature, TBST is used for washing membranes, a gel imaging system is used for exposure imaging, and the result is shown in fig. 3 and 4, and as can be seen from fig. 3 and 4, the monoclonal antibody can be combined with human CD14 antigen.
1.5 Cloning of antibody light and heavy chain variable regions
Extracting RNA of 2D11 and 3G3 hybridoma cells, synthesizing cDNA by using takara reverse transcription kit, and performing PCR (pre-denaturation at 94 ℃ for 5 min) by using the degenerate primer of the mouse antibody gene as a template; (94 ℃ C. 90s,50 ℃ C. 90s,72 ℃ C. 2 min) 35cycles,72 ℃ C. 10min,4 ℃ C. Preservation ]; the PCR product was recovered by gel ligation into pMD18-T vector, transformed into E.coli JM109, and positive clones were picked for sequencing identification. The amino acid sequence of the sCD14/2D11 light chain variable region is shown as SEQ ID No.2, the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.3, the amino acid sequence of the sCD14/3G3 light chain variable region is shown as SEQ ID No.4, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 5.
2. HRP-labeled sCD14/3G3 monoclonal antibody
10Mg of sCD14/3G3 monoclonal antibody is taken, put into a dialysis bag and placed in carbonate buffer solution (1.59G/L Na 2CO3; 2.93g/L NaHCO3) with pH=9.4 for dialysis; weighing 20mg of HRP (purchased from national drug group), dissolving with 1mL of ultrapure water, weighing 128mg of NaIO 4, dissolving with 5mL of ultrapure water, adding 0.8mL of NaIO 4 solution into the HRP solution, and standing at 4 ℃ for 30min; preparing a 10% glycol solution, adding 160 mu L of the 10% glycol solution into the HRP+NaIO 4 mixed solution, and standing for 30min at room temperature in a dark place; adding the mixed solution (HRP+NaIO 4 +ethylene glycol) into the sCD14/3G3 antibody under dialysis, and continuing dialysis at 4 ℃ in the dark for overnight; preparing a sodium borohydride solution with the concentration of 0.5wt%, taking out the antibody in the dialysis bag, adding 320 mu L of the sodium borohydride solution into the antibody solution, and placing the solution at the temperature of 4 ℃ in a dark place for 2 hours; adding an equal volume of saturated ammonium sulfate, and standing at 4 ℃ for 2 hours; centrifuging at 4deg.C, collecting precipitate, dissolving in PBS, loading into dialysis bag, dialyzing in PBS buffer solution overnight, taking out labeled antibody in dialysis bag, and preserving at-20deg.C in 50% glycerol.
3. Human soluble CD14 detection by sCD14/2D11 and HRP-sCD 14/3G3 double antibody sandwich method
Carbonate buffer (1.59 g/L Na 2CO3; 2.93g/L NaHCO3) coated 100. Mu.L of 1.25. Mu.g/ml sCD14/2D11 into polystyrene Elisa assay wells, and allowed to stand overnight at 4 ℃; PBST solution (8 g/L NaCl; 5.8g/L Na 2HPO4; 0.6g/L NaH2PO4; 0.5%Tween 20) was washed 3 times; mu.L of PBST+2% BSA (blocking solution) was added and blocked at 37℃for 1 hour; washing with PBST solution for 3 times; add 100. Mu.L of gradient concentration of CD14 antigen standard (10 ng/mL, 8ng/mL, 6ng/mL, 4ng/mL, 2ng/mL, 1ng/mL, 0.5 ng/mL) and incubate at 37℃for 1h; washing with PBST solution for 3 times; HRP-sCD 14/3G3 diluted (1:1000) with 100. Mu.L of blocking solution was added and incubated for 1h at 37 ℃; washing with PBST solution for 5 times; adding TMB chromogenic substrate, adding sulfuric acid stop solution after 5min to stop reaction, measuring OD 450 by using an enzyme-labeling instrument, and drawing a standard curve which is shown in FIG. 5. As can be seen from fig. 5, the linear correlation coefficient of the standard curve is 0.999, and the linearity of the detection interval is good.
4. Detection of soluble CD14 in human serum by sCD14/2D11 and HRP-sCD 14/3G3 double antibody sandwich method
Carbonate buffer coated 100. Mu.L of 1.25. Mu.g/mL sCD14/2D11 into polystyrene Elisa assay wells, standing overnight at 4 ℃; washing with PBST solution for 3 times; 300. Mu.L of PBST+2% BSA was added and blocked at 37℃for 1 hour; washing with PBST solution for 3 times; 100 mu L of standard substances (10 ng/mL, 8ng/mL, 6ng/mL, 4ng/mL, 2ng/mL, 1ng/mL and 0.5 ng/mL) with gradient concentration are added into a standard substance detection hole, a diluted serum sample is added into a sample detection hole, and the mixture is incubated for 1h at 37 ℃; washing with PBST solution for 3 times; HRP-sCD 14/3G3 diluted (1:1000) with 100. Mu.L of blocking solution was added and incubated for 1h at 37 ℃; washing with PBST solution for 5 times; adding TMB chromogenic substrate, adding stop solution after 5min, measuring OD 450 by using an enzyme-labeled instrument, drawing a standard curve, and calculating the concentration of soluble CD14 in serum by using a standard curve formula.
5. Detection of soluble CD14 in serum of patients with bacterial infection and patients with liver cancer by the method
5.1 Serum was collected from 96 bacterial infected patients (positive for microbial detection) and 105 healthy controls, and the concentration of soluble CD14 in the serum was measured, and as shown in fig. 6, the concentration of soluble CD14 in the serum of infected patients was significantly higher than that in healthy controls (P < 0.001).
5.2 Serum was collected from 24 liver cancer patients and 105 healthy controls, and the concentration of soluble CD14 in the serum was measured, and as shown in fig. 7, the concentration of soluble CD14 in the serum of liver cancer patients was significantly higher than that of healthy controls (P < 0.001).
5.3 The diagnostic efficacy of human serum soluble CD14 concentration on infected patients was determined by ROC curve analysis, and the results are shown in fig. 8, with specificity=91.4% and sensitivity=71.9% of diagnosis, indicating that the detection method of the present invention can be applied to clinical diagnosis of infected patients.
5.4 The diagnostic efficacy of human serum soluble CD14 concentration on liver cancer is determined by using ROC curve analysis, and the result is shown in figure 9, the specificity of diagnosis is=96.2%, and the sensitivity is=87.5%, which indicates that the detection method can be applied to clinical diagnosis of liver cancer patients.
Therefore, the sCD14/2D11 and HRP-sCD 14/3G3 provided by the invention can be used for detecting human soluble CD14 by using a double antibody sandwich method, and can be used for preparing a human soluble CD14 detection kit.
Claims (8)
1. A monoclonal antibody that specifically binds to human CD14, characterized in that: comprising two antibodies, sCD14/2D11 and sCD14/3G3, respectively, each of said sCD14/2D11 and sCD14/3G3 being capable of recognizing and binding to the polypeptide chain of the amino acid sequence of CD 14;
The amino acid sequence of the light chain variable region of sCD14/2D11 is shown as SEQ ID No.2, and the amino acid sequence of the heavy chain variable region of sCD14/2D11 is shown as SEQ ID No. 3;
the amino acid sequence of the light chain variable region of sCD14/3G3 is shown as SEQ ID No.4, and the amino acid sequence of the heavy chain variable region of sCD14/3G3 is shown as SEQ ID No. 5.
2. The monoclonal antibody that specifically binds to human CD14 according to claim 1, characterized in that: the amino acid sequence of the CD14 is shown as SEQ ID No. 1.
3. A monoclonal antibody that specifically binds to human CD14, characterized in that: comprising two antibodies, sCD14/2D11 and HRP-sCD14/3G3, respectively, each of said sCD14/2D11 and HRP-sCD14/3G3 being capable of recognizing and binding to the polypeptide chain of the amino acid sequence of CD 14;
The HRP-sCD14/3G3 is HRP-marked sCD14/3G3;
The amino acid sequence of the light chain variable region of sCD14/2D11 is shown as SEQ ID NO. 2, and the amino acid sequence of the heavy chain variable region of sCD14/2D11 is shown as SEQ ID NO. 3;
The amino acid sequence of the light chain variable region of sCD14/3G3 is shown as SEQ ID NO. 4, and the amino acid sequence of the heavy chain variable region of sCD14/3G3 is shown as SEQ ID NO. 5.
4. A monoclonal antibody which specifically binds to human CD14 according to claim 3, characterized in that: the amino acid sequence of the CD14 is shown as SEQ ID NO. 1.
5. Use of a monoclonal antibody according to claim 3 or 4 which specifically binds human CD14 for the preparation of a diagnostic reagent for the detection of human soluble CD 14.
6. The use according to claim 5, characterized in that: the diagnostic reagent for detecting human soluble CD14 is a diagnostic reagent for detecting soluble CD14 in human serum or plasma.
7. The use according to claim 5, characterized in that: the diagnostic reagent for detecting human soluble CD14 is used for preparing a detection reagent for infectious diseases or a detection reagent for liver cancer.
8. The use according to claim 7, characterized in that: the infectious disease is a bacterial infection.
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| CN118290585A (en) * | 2024-06-04 | 2024-07-05 | 苏州为度生物技术有限公司天津分公司 | Anti-human CD14 engineering antibody and application thereof |
| CN118620079A (en) * | 2024-08-12 | 2024-09-10 | 苏州欣协生物科技有限公司 | Anti-human CD14 antibody, amino acid sequence and nucleotide sequence thereof and application thereof |
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| CN118290585A (en) * | 2024-06-04 | 2024-07-05 | 苏州为度生物技术有限公司天津分公司 | Anti-human CD14 engineering antibody and application thereof |
| CN118290585B (en) * | 2024-06-04 | 2024-08-30 | 苏州为度生物技术有限公司天津分公司 | Anti-human CD14 engineering antibody and application thereof |
| CN118620079A (en) * | 2024-08-12 | 2024-09-10 | 苏州欣协生物科技有限公司 | Anti-human CD14 antibody, amino acid sequence and nucleotide sequence thereof and application thereof |
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