CN114891752B - Hybridoma cell strain of anti-S-adenosyl homocysteine monoclonal antibody and application - Google Patents
Hybridoma cell strain of anti-S-adenosyl homocysteine monoclonal antibody and application Download PDFInfo
- Publication number
- CN114891752B CN114891752B CN202210515653.8A CN202210515653A CN114891752B CN 114891752 B CN114891752 B CN 114891752B CN 202210515653 A CN202210515653 A CN 202210515653A CN 114891752 B CN114891752 B CN 114891752B
- Authority
- CN
- China
- Prior art keywords
- hybridoma cell
- adenosyl homocysteine
- monoclonal antibody
- cell strain
- sah
- 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.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
- C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/02—Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion
-
- 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
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6806—Determination of free amino acids
- G01N33/6812—Assays for specific amino acids
- G01N33/6815—Assays for specific amino acids containing sulfur, e.g. cysteine, cystine, methionine, homocysteine
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
- C12N2510/02—Cells for production
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to a hybridoma cell strain secreting an anti-S-adenosyl homocysteine monoclonal antibody and application thereof, belonging to the technical field of immunological disease diagnosis. The hybridoma cell strain is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms (China) for 03 months in 2022, the preservation address is the North-West-Lu No. 1 and No. 3 of the Korean region of Beijing, the preservation number is CGMCC No.45110, and the hybridoma cell strain is classified and named as a monoclonal cell strain. The S-adenosyl homocysteine monoclonal antibody secreted by the hybridoma cell strain has better sensitivity (IC) to SAH 50 The value is 2.9 nmol/L) and specificity (the cross reaction rate to analogues is less than 1%), can realize the high-sensitivity detection of SAH content in human serum, provides raw materials for the immunological detection of the SAH content in human body, and can play a role in effectively monitoring cardiovascular and cerebrovascular diseases.
Description
Technical Field
The invention relates to the technical field of immunological disease diagnosis, in particular to a hybridoma cell strain secreting an anti-S-adenosyl homocysteine monoclonal antibody and application thereof.
Background
S-adenosyl homocysteine (S-adenosyl homocysteine, SAH), also known as S-adenosyl homocysteine. Methionine in human body is first combined with ATP to produce S-adenosylmethionine (S-adenosyl methionine, SAM) under the action of adenyltransferase in the metabolic process, SAM is demethylated under the catalysis of methyltransferase to produce S-adenosylhomoSAH. SAH is in turn an important precursor for homocysteine (Hcy) and can be reversibly hydrolysed to Hcy and adenosine by SAH hydrolase (S-adenosyl homocysteine hydrolase, SAHH). It has been shown that elevated SAH levels can induce vascular endothelial cell damage, a mechanism associated with inhibition of DNA methylation processes. Plasma SAH levels can be an important predictor of the risk of cardiovascular disease in coronary angiographic patients. And has research to prove the feasibility of SAH as an ischemic cerebral apoplexy screening sensitivity index and a therapeutic target.
At present, the main detection method of the S-adenosyl homocysteine content is a High Performance Liquid Chromatography (HPLC) method, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, a fluorescence polarization immunoassay (FPLA) method and the like. All the detection methods involve complicated instruments and equipment, special operation is needed, steps are tedious and time-consuming, and on-site rapid detection is difficult to realize. The detection method based on the immunological principle, such as enzyme-linked immunosorbent assay (ELISA) and Lateral Flow Immunochromatography (LFIA) test strip method, has the characteristics of simple method and operation, time and labor saving and capability of rapidly detecting the target object on site. The immunological method uses antigen and antibody as raw material to detect target antigen in sample to be detected, so that it is critical to prepare monoclonal antibody with high sensitivity and strong specificity. At present, the SAH has fewer immunological detection methods, and monoclonal antibody IC is available on the market 50 About 2400nmol/L, and has certain cross to SAM, it is difficult to reach the requirement of high sensitivity, high specificity detection.
Disclosure of Invention
Therefore, the invention aims to solve the technical problems of low immunological detection sensitivity and poor specificity of SAH in the prior art.
In order to solve the technical problems, the invention provides a hybridoma cell strain of an anti-S-adenosyl homocysteine monoclonal antibody and application thereof. The invention obtains the monoclonal antibody hybridoma cell strain with higher sensitivity and specificity to S-adenosyl homocysteine (SAH) through the steps of cell fusion, HAT selective medium culture, cell supernatant screening by ic-ELISA, subcloning and the like of the prepared S-adenosyl homocysteine complete antigen immune animal.
The first object of the present invention is to provide a hybridoma cell strain secreting anti-S-adenosyl homocysteine monoclonal antibody, wherein the hybridoma cell strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on the day of 2022, the preservation address is No. 3 of North Chen West Lu No. 1 in the Korean region of Beijing city, the preservation number is CGMCC No.45110, and the hybridoma cell strain obtained by the present invention belongs to the monoclonal cell strain.
The second object of the present invention is to provide a method for producing a hybridoma cell strain secreting an anti-S-adenosyl homocysteine monoclonal antibody, comprising the steps of,
(1) S-adenosyl homocysteine hapten is used for preparing S-adenosyl homocysteine complete antigen, and the obtained S-adenosyl homocysteine complete antigen is prepared into antigen-containing Freund complete adjuvant and antigen-containing Freund incomplete adjuvant;
(2) Performing primary immunization, booster immunization and sprint immunization on the immunized animal, wherein the primary immunization adopts the antigen-containing Freund complete adjuvant in the step (1), the booster immunization adopts the antigen-containing Freund incomplete adjuvant in the step (1), and the sprint immunization adopts the S-adenosyl homocysteine complete antigen in the step (1);
(3) And (3) taking spleen cells and myeloma cells of the immunized animal subjected to sprint immunization in the step (2) for cell fusion, and obtaining the hybridoma cell strain.
In one embodiment of the present invention, in step (1), the S-adenosyl homocysteine hapten has the following structural formula:
in one embodiment of the invention, in step (1), the S-adenosyl homocysteine complete antigen is obtained by coupling to a carrier protein after activation of the S-adenosyl homocysteine hapten.
In one embodiment of the invention, the preparation method of the S-adenosyl homocysteine complete antigen comprises the following steps of dissolving the S-adenosyl homocysteine hapten and N, N' -carbonyl diimidazole in an organic solvent, reacting to obtain a mixed solution, and then adding the mixed solution into a Keyhole Limpet Hemocyanin (KLH) solution to react to obtain the S-adenosyl homocysteine complete antigen.
In one embodiment of the invention, the organic solvent is dimethyl sulfoxide (DMSO).
In one embodiment of the invention, the carrier protein solution is diluted with a carbonate buffer solution CBS.
In one embodiment of the invention, the molar ratio of S-adenosyl homocysteine hapten, N' -carbonyldiimidazole and carrier protein is 6000:60:1.
in one embodiment of the present invention, in the step (1), the preparation method of the complete antigen of S-adenosyl homocysteine comprises the following steps of dissolving the complete antigen of S-adenosyl homocysteine (SAH) and N, N' -Carbonyldiimidazole (CDI) in dimethyl sulfoxide (DMSO), and stirring to react to obtain solution A; diluting Keyhole Limpet Hemocyanin (KLH) with Carbonate Buffer Solution (CBS) to obtain solution B; adding the solution A into the solution B for reaction to obtain a reaction solution; and (3) dialyzing the reaction solution by using Phosphate Buffer (PBS) to obtain the S-adenosyl homocysteine complete antigen.
In one embodiment of the invention, in step (2), the entire immunization process comprises 1 first immunization, 3-5 booster immunizations, and 1 sprint immunization.
In one embodiment of the invention, in step (2), the interval between the first immunization and the boost is 28-31 days, the interval between the boost is 20-22 days, and the interval between the boost and the sprint is 18-21 days during the whole immunization process.
In one embodiment of the present invention, in the step (2), the immune animals are collected during the booster immunization, the serum immune titer and the immune suppression capacity of the immune animals are detected by ic-ELISA, the immune animals with high titer and high suppression rate are screened, and the screened immune animals are boosted with the incomplete Freund adjuvant containing antigen for the last two times.
In one embodiment of the invention, the blood collection is performed on days 6-8 after the end of the booster immunization procedure.
In one embodiment of the invention, in step (2), the first immunization and booster immunization is injected subcutaneously into the immunized animal via the back.
In one embodiment of the invention, in step (2), the sprint immunity is injected intraperitoneally into the immunized animal.
In one embodiment of the present invention, in step (3), the cell fusion is to culture the fused cells in a medium, detect positive cells by using ic-ELISA, and further determine the inhibition effect of positive cells by using ic-ELISA, subcloning the positive cells with the best inhibition by limiting dilution, thereby obtaining hybridoma cell lines.
In one embodiment of the invention, the medium is RPMI-1640 medium.
In one embodiment of the invention, the number of subclones is 2-4.
In one embodiment of the invention, in step (3), the cell fusion is performed by conventional PEG methods.
In one embodiment of the invention, in step (3), the cell fusion is performed 2-4 days after the termination of the sprint immunization.
In one embodiment of the invention, a method for preparing a hybridoma cell strain secreting an anti-S-adenosyl homocysteine monoclonal antibody specifically comprises the following steps,
(1) S-adenosyl homocysteine hapten is used for preparing S-adenosyl homocysteine complete antigen, and the obtained S-adenosyl homocysteine complete antigen is prepared into antigen-containing Freund complete adjuvant and antigen-containing Freund incomplete adjuvant;
(2) Injecting the obtained complete Freund adjuvant containing the antigen into an immunized animal body through subcutaneous injection on the back for multiple times, wherein the complete Freund adjuvant containing the antigen is adopted for the first immunization, and the incomplete Freund adjuvant containing the antigen is adopted for the booster immunization;
(3) Collecting blood of the mice subjected to the immunization process, detecting serum immune titer and immune suppression capacity of the mice by indirect ELISA, and screening the mice with high S-adenosyl homocysteine antibody content in the serum to obtain immunity;
(4) The screened mice are subjected to final two times of boosting immunization by using Freund incomplete adjuvant containing antigen, and then are subjected to sprint immunization by intraperitoneal injection, wherein the sprint immunization is performed by using S-adenosyl homocysteine complete antigen without Freund adjuvant;
(5) Fusing spleen cells and myeloma cells of mice subjected to sprint immunization, culturing the fused cells through a culture medium, detecting positive cell holes by using an ic-ELISA (enzyme-linked immunosorbent assay), further measuring the inhibition effect of the positive cell holes by using the ic-ELISA method, subcloning the cell holes with high positive value, high inhibition rate and minimum cell mass number by using a limiting dilution method, and finally screening out hybridoma cell strains capable of secreting the anti-S-adenosyl homocysteine monoclonal antibody.
The third object of the invention is to provide an application of the hybridoma cell strain in preparing an S-adenosyl homocysteine monoclonal antibody.
The fourth object of the invention is to provide an S-adenosyl homocysteine monoclonal antibody which is secreted by a hybridoma cell strain with the preservation number of CGMCC No.45110.
In one embodiment of the invention, paraffin oil is injected into the abdominal cavity of an immunized animal, then hybridoma cell strain with the preservation number of CGMCC No.45110 is injected into the abdominal cavity, ascites is collected after injection, and the ascites is purified, so that the S-adenosyl homocysteine monoclonal antibody is obtained for low-temperature preservation.
In one embodiment of the invention, 8-10 week old BALB/c mice are intraperitoneally injected with 1mL of paraffin oil, 1X 10 per mouse after 7 days 6 Collecting ascites from the 7 th day of hybridoma cell strain with the preservation number of CGMCC No.45110, purifying the ascites by an octanoic acid-ammonium sulfate method, and preserving the obtained S-adenosyl homocysteine monoclonal antibody at the temperature of minus 20 ℃.
A fifth object of the present invention is to provide a composition comprising said hybridoma cell line and/or said S-adenosyl homocysteine monoclonal antibody.
A sixth object of the present invention is to provide a kit comprising one or more of the hybridoma cell line, the S-adenosyl homocysteine monoclonal antibody and the composition.
The seventh object of the present invention is to provide a test strip comprising one or more of the hybridoma cell line, the S-adenosyl homocysteine monoclonal antibody, the composition and the kit.
The eighth object of the invention is to provide an application of the hybridoma cell strain, the S-adenosyl homocysteine clone antibody, the composition, the kit or the test strip in detection of S-adenosyl homocysteine, in particular to detection of SAH content in human serum.
Compared with the prior art, the technical scheme of the invention has the following advantages:
the S-adenosyl homocysteine monoclonal antibody secreted by the hybridoma cell strain has better sensitivity (IC) to SAH 50 The value is 2.9 nmol/L) and specificity (the cross reaction rate to analogues is less than 1%), can realize the high-sensitivity detection of SAH content in human serum, provides raw materials for the immunological detection of the SAH content in human body, can play a role in effectively monitoring cardiovascular and cerebrovascular diseases, and has practical application value.
Preservation of biological materials
The hybridoma cell strain secreting the anti-S-adenosyl homocysteine monoclonal antibody belongs to a monoclonal cell strain, the hybridoma cell strain is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on the 03 th day of 2022, the preservation address is the North Chen West Lu No. 1, 3 of the Korean region of Beijing city, the preservation number is CGMCC No.45110, and the hybridoma cell strain is classified and named as the monoclonal cell strain.
Drawings
In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, in which:
FIG. 1 is a standard curve of the inhibition of S-adenosyl homocysteine by the S-adenosyl homocysteine monoclonal antibody of the invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
(1) The following examples relate to the following media:
RPMI-1640 medium (mg/L): l-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cystine dihydrochloride 65.15, L-glutamic acid 20, glycine 10, L-histidine 15, L-hydroxyproline 20, L-isoleucine 50, L-leucine 50, L-lysine hydrochloride 40, L-methionine 15, L-phenylalanine 15, L-proline 20, L-serine 30, L-threonine 20, L-tryptophan 5, L-tyrosine 23.19, L-valine 20, p-aminobenzoic acid 1, calcium nitrate 100, anhydrous magnesium sulfate 48.84, anhydrous sodium dihydrogen phosphate 676.13, potassium chloride 400, sodium chloride 6000, glucose 2000, reduced glutathione 1, phenol red 5, L-glutamine 300, biotin 0.2, D-calcium pantothenate 0.25, folic acid 1, i-inositol 35, nicotinamide 1, choline chloride 3, pyridoxine hydrochloride 1, riboflavin 0.2, thiamine hydrochloride 1, vitamin B120.005, sodium bicarbonate 2000.
(2) The reagents involved in the following examples were as follows:
carbonate Buffer (CBS): weighing Na 2 CO 3 1.59g，NaHCO 3 2.93g, respectively dissolving in a small amount of double distilled water, mixing, adding double distilled water to about 800mL, mixing, adjusting pH to 9.6, adding double distilled water to 1000mL, and storing at 4deg.C for use;
phosphate Buffer (PBS): 8.00g NaCl,0.2g KCl,0.2g KH 2 PO 4 ，2.9g Na 2 HPO 4 ·12H 2 O is dissolved in 800mL of pure water, pH is regulated to 7.2-7.4 by NaOH or HCl, and volume is regulated to 1000mL;
washing solution (PBST): 1000mL of 0.01mol/L PBS solution with pH 7.4 is added with 0.5mL of Tween-20;
PBST: PBS containing 0.05% Tween-20;
antibody dilution: a wash buffer containing 0.1% gelatin;
TMB color development liquid: liquid A：Na 2 HPO 4 ·12H 2 18.43g of O, 9.33g of citric acid and pure water to 1000mL; and (2) liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. The volume ratio of the solution B is 5:1 to obtain TMB color development liquid, and mixing immediately.
(3) The detection method involved in the following examples is as follows:
the detection method of the S-adenosyl homocysteine inhibition rate comprises the following steps: the most appropriate antigen and antibody concentrations in the ic-ELISA were selected by a checkerboard assay. The antigen was diluted to 1,0.3,0.1 and 0.03 μg/mL with Carbonate Buffer (CBS) and the antibody was diluted to 1,0.3,0.1 and 0.03 μg/mL with antibody dilution. After selecting the optimal working point, diluting the S-adenosyl homocysteine standard substance by 80, 26.7,8.9,3.0,1.0,0.3,0.1 and 0ng/mL, and calculating IC according to the IC-ELISA operation steps, finally, using OriginPro 8.5 as a graph to obtain the S-adenosyl homocysteine standard inhibition curve 50 。
Examples
A hybridoma cell strain secreting an anti-S-adenosyl homocysteine monoclonal antibody and a preparation method thereof specifically comprise the following steps:
A. preparation of complete antigen
Preparation of the immunogen SAH-CDI-KLH: 2.56mg of SAH and 10.81mg of N, N' -Carbonyldiimidazole (CDI) were weighed, dissolved in 500. Mu.L of DMSO solution, and reacted at 37℃with stirring for 2 hours, designated as solution A; 5mg of KLH was taken and the volume was set to 2mL with 0.05M carbonate buffer, designated as solution B. Solution a was slowly added dropwise to solution B and the reaction was stirred at room temperature for 24h. The reacted solution was dialyzed against 0.01M phosphate buffer for 3d at-20℃until use.
B. Preparation of coating Material
1.72mg BSA,2.56mg EDC and 1.54mg of NHS are weighed and dissolved in 500 mu L of DMSO solution, and stirred at room temperature for reaction for 2 hours, and then marked as solution C; 5mg of BSA was weighed out and dissolved in 2mL of 0.05M carbonate buffer, designated as solution D. Solution C was slowly added dropwise to solution D and the reaction was stirred at room temperature for 6h. The reacted solution was dialyzed against 0.01M phosphate buffer for 3d at-20℃until use.
C. Immunization of mice
Healthy 6-8 week old BALB/c mice were selected for immunization. The immunogen SAH-CDI-KLH is mixed and emulsified with the equivalent Freund's adjuvant to obtain injection, and BALB/c mice are immunized by subcutaneous multipoint injection on the neck and back. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the subsequent immunization. The interval between the first immunization and the second boost was 28 days, and the interval between the multiple boosts was 21 days. 7 days after the third immunization (mice were bled 5 μl+995 μl of antibody diluent = 200-fold diluted antiserum), titers and inhibition of the mouse serum were determined using ic-ELISA, mice with high titers and high inhibition were selected, and 21 days after the fifth immunization, i.e. the immunization was given by intraperitoneal injection of immunogen, requiring halving of the dose of the sprint immunization and no adjuvant.
D. Cell fusion
After three days of sprint immunization, cell fusion was performed according to the conventional PEG method, specifically as follows:
(a) Immediately after the mice were sacrificed, they were sterilized in 75% alcohol, soaked for about 5min, the spleens of the mice were removed by aseptic manipulation, spleen cell suspensions were moderately ground and obtained by passing through a 200 mesh cell screen, collected, centrifuged (1200 rpm,8 min), the spleen cells were washed three times with RPMI-1640 medium, and the tissue components from which non-cells were discarded were selected. After the last centrifugation, diluting spleen cells to a certain volume, and counting for later use;
(b) Collecting murine myeloma SP2/0 cells: SP2/0 tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) 7-10 days before fusion. The number of SP2/0 tumor cells required before fusion reaches (1-4). Times.10 7 The SP2/0 cells are guaranteed to be in the logarithmic growth phase before fusion. During fusion, collecting tumor cells, suspending in RPMI-1640 basic culture solution, and performing cell count;
(c) The fusion process was 7min. 1min, 1mL of PEG is added dropwise to the cells in the centrifuge tube from slow to fast, and the centrifuge tube is shaken during the period (the same is true for the subsequent dropping process); and (2) standing for 2 min. Dripping 1mL of RPMI-1640 culture medium in the period of 1min for 3min and 4 min; dripping 2mL of RPMI-1640 culture medium in the period of 1min at the 5 th and 6 th min; at 7min, 1mL of RPMI-1640 medium was added dropwise every 10 s. Then the temperature bath is carried out at 37 DEG C5min. Centrifugation (800 rpm,8 min), discarding supernatant, re-suspending in RPMI-1640 screening medium containing 20% fetal bovine serum, 2% HAT, adding 200. Mu.L/well to 96 well cell plates, and placing at 37℃and 5% CO 2 Culturing in an incubator.
E. Cell screening and cell strain establishment
The cells were subjected to half-replacement of the RPMI-1640 selection medium on day 3 of cell fusion, full replacement with a 100 XHT RPMI-1640 transition medium containing 20% fetal bovine serum and 1% on day 5, and cell supernatants were collected on day 7 for selection. Screening is carried out in two steps: the first step is to screen out positive cell holes by using ic-ELISA, and the second step is to measure the inhibition effect of positive cell holes by using ic-ELISA by using SAH as a standard substance. Cell holes with better SAH inhibition are selected, subcloning is carried out by a limiting dilution method, and detection is carried out by the same method. After three subcloning, monoclonal hybridoma cell lines were obtained.
Test case
A. Preparation of monoclonal antibodies
Taking 8-10 week old BALB/c mice, and injecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; intraperitoneal injection of 1X 10 per mouse after 7 days 6 Hybridoma cells, ascites was collected from the seventh day, and the ascites was purified by the octanoic acid-ammonium sulfate method. And (3) desalting by 0.01M PBS (phosphate buffered saline) dialysis, and finally obtaining the purified monoclonal antibody, and storing at-20 ℃.
IC measured by IC-ELISA 50 The value is 2.9nmol/L, which shows that the antibody has higher sensitivity to SAH; and verifying the cross reaction of the S-adenosyl homocysteine analogue with respect to the SAM analogue, wherein the cross rate of the S-adenosyl homocysteine analogue is less than 1%, and the cross rate is = (IC of S-adenosyl homocysteine) 50 IC of analog 50 ) By 100%, the monoclonal antibodies obtained by the present invention have higher specificity and sensitivity to SAH according to the cross-reactivity values, as shown in Table 1.
Table 1 shows the IC of monoclonal antibodies to SAH, SAM, adenosine, hcy, cysteine, cystathionine 50 Cross-reaction rate:
TABLE 1
| IC 50 (nmol/L) | Cross reaction rate | |
| SAH | 2.9 | 100％ |
| SAM | >1000 | <1％ |
| Adenosine | >1000 | <1％ |
| Hcy | >1000 | <1％ |
| Cysteine (S) | >1000 | <1％ |
| Cystathionine (cystathionine) | >1000 | <1％ |
B. Identification of monoclonal antibodies
The monoclonal antibody prepared from the hybridoma cell strain through in-vivo ascites is applied to an S-adenosyl homocysteine addition recovery test, and the specific steps are as follows:
(a) Coating: the coating raw SAH-EDC-BSA was diluted 3-fold with 0.05M carbonate buffer starting at 1. Mu.g/mL, 100. Mu.L/well, and reacted at 37℃for 2 hours.
(b) Washing: the solution in the plate was thrown off and washed 3 times with washing liquid for 3min each time.
(c) Closing: after drying, 200. Mu.L/well of blocking solution was added thereto and reacted at 37℃for 2 hours. And (5) drying for standby after washing.
(d) Sample adding: the monoclonal antibodies were diluted 3-fold in gradient from 1. Mu.g/mL with antibody dilutions and added to the coated wells at each dilution, 50. Mu.L/well; then sequentially adding diluted SAH standard substances (corresponding cross-product standard substances are added when cross detection is carried out), and reacting for 30min at 37 ℃; after taking out the decanted liquid and washing thoroughly, add 1: 3000-fold dilution of HRP-goat anti-mouse IgG, 100. Mu.L/well, and reaction at 37℃for 30min.
(e) Color development: and taking out the ELISA plate, pouring out the liquid in the holes, fully washing, adding 100 mu L of TMB color developing solution into each hole, and carrying out light-shielding reaction for 15min at 37 ℃.
(f) Termination and measurement: 50. Mu.L of stop solution was added to each well to terminate the reaction, and the OD450 value of each well was measured by using a microplate reader.
The standard curve of S-adenosyl homocysteine inhibition by S-adenosyl homocysteine monoclonal antibody is shown in FIG. 1, and IC of S-adenosyl homocysteine monoclonal antibody can be measured by IC-ELISA 50 The value is 2.9nmol/L, which shows that the antibody has better sensitivity to S-adenosyl homocysteine, and can be used for the analysis and detection of S-adenosyl homocysteine.
Comparative example
Compared with the existing or previous products on the market, the invention makes certain improvement and innovation on the antigen preparation method, and the prepared products have greatly improved properties, such as:
in contrast to Hao Xiujuan et al, 2014 (CN 104479022 a), both the immunogen and the coating were prepared by EDC, identical to the binding site of the antibody.IC of finally obtained monoclonal antibody 301 50 The value was 547nmol/L and the cross-reaction rate with SAM was 1.69%.
The antibody sensitivity was about 2400nmol/L compared with the Abcam rabbit monoclonal antibody, which had a cross-reactivity of 1.41% with SAM.
The invention synthesizes immunogen by adopting CDI method coupled hydroxyl, which better exposes active group amino and carboxyl of SAH, thus ensuring immunogenicity; the EDC method is adopted to synthesize the coating raw material as the coating, and the competitive binding site of the coating raw material is different from that of the antibody, so that the competitive inhibition capability of the SAH standard product is effectively improved. The monoclonal antibody IC obtained 50 The value is 2.9nmol/L, and the cross reaction rate with SAM and the like is lower than 0.2 percent. The sensitivity is improved by 200-800 times, and the specificity is improved by 5-10 times.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (6)
1. The hybridoma cell strain secreting the anti-S-adenosyl homocysteine monoclonal antibody is characterized in that the hybridoma cell strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the year of 2022 and 03, and the preservation address is the number 3 of North Chen West Lu 1 in the Korean region of Beijing city, and the preservation number is CGMCC No.45110.
2. The use of the hybridoma cell line of claim 1 for preparing an S-adenosyl homocysteine monoclonal antibody.
3. An S-adenosyl homocysteine monoclonal antibody, which is secreted by the hybridoma cell line of claim 1.
4. A composition comprising the hybridoma cell line of claim 1 and/or the S-adenosyl homocysteine monoclonal antibody of claim 3.
5. A kit comprising one or more of the hybridoma cell line of claim 1, the S-adenosyl homocysteine monoclonal antibody of claim 3, and the composition of claim 4.
6. A test strip comprising one or more of the hybridoma cell line of claim 1, the S-adenosyl homocysteine monoclonal antibody of claim 3, and the composition of claim 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210515653.8A CN114891752B (en) | 2022-05-12 | 2022-05-12 | Hybridoma cell strain of anti-S-adenosyl homocysteine monoclonal antibody and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210515653.8A CN114891752B (en) | 2022-05-12 | 2022-05-12 | Hybridoma cell strain of anti-S-adenosyl homocysteine monoclonal antibody and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114891752A CN114891752A (en) | 2022-08-12 |
| CN114891752B true CN114891752B (en) | 2023-06-02 |
Family
ID=82722062
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210515653.8A Active CN114891752B (en) | 2022-05-12 | 2022-05-12 | Hybridoma cell strain of anti-S-adenosyl homocysteine monoclonal antibody and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114891752B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101246177A (en) * | 2007-02-14 | 2008-08-20 | 北京华安佛医药研究中心有限公司 | Homo-type cysteine immunity colloidal gold detecting test paper and method for producing the same |
| CN104479022A (en) * | 2014-10-23 | 2015-04-01 | 湖南天合生物技术有限公司 | Anti-S-adenosyl-L-homocysteine monoclonal antibody 301, hybridomas thereof, composition, colloidal gold test paper, kit and uses |
| CN104597007A (en) * | 2014-11-30 | 2015-05-06 | 陈燕婷 | Homocysteine kit based on aptamer fluorescence probe HCy5 and detection method thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050019937A1 (en) * | 2002-11-29 | 2005-01-27 | Industrial Technology Research Institute | Assay and kit for homocysteine |
| US9885723B2 (en) * | 2013-03-15 | 2018-02-06 | Hunan Skyworld Biotechnologies Co | Immunoassays of S-adenosylmethionine and methylation index in personalized medicine and health evaluation |
| US9958447B2 (en) * | 2013-03-15 | 2018-05-01 | Hunan Skyworld Biotechnologies Co | Immunoassay of S-adenosylmethionine in personalized medicine and health or cancer evaluation |
-
2022
- 2022-05-12 CN CN202210515653.8A patent/CN114891752B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101246177A (en) * | 2007-02-14 | 2008-08-20 | 北京华安佛医药研究中心有限公司 | Homo-type cysteine immunity colloidal gold detecting test paper and method for producing the same |
| CN104479022A (en) * | 2014-10-23 | 2015-04-01 | 湖南天合生物技术有限公司 | Anti-S-adenosyl-L-homocysteine monoclonal antibody 301, hybridomas thereof, composition, colloidal gold test paper, kit and uses |
| CN104597007A (en) * | 2014-11-30 | 2015-05-06 | 陈燕婷 | Homocysteine kit based on aptamer fluorescence probe HCy5 and detection method thereof |
Non-Patent Citations (1)
| Title |
|---|
| Development of a colloidal gold strip assay for the detection of total homocysteine in serum samples;Beijun Ang等;Analyst;第147卷;第4348-4355页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114891752A (en) | 2022-08-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113736744B (en) | Digitoxin monoclonal antibody hybridoma cell strain and application thereof | |
| CN113621583B (en) | Hybridoma cell strain secreting dimethomorph monoclonal antibody and application thereof | |
| CN114107219B (en) | Hybridoma cell strain secreting insecticidal amidine monoclonal antibody and application thereof | |
| CN114891752B (en) | Hybridoma cell strain of anti-S-adenosyl homocysteine monoclonal antibody and application | |
| CN114836387B (en) | 11-alpha hydroxyprogesterone monoclonal antibody hybridoma cell strain and application thereof | |
| CN114181911B (en) | Hybridoma cell strain secreting spirolactone and metabolite monoclonal antibody thereof and application of hybridoma cell strain | |
| CN111763658A (en) | Hybridoma cell strain secreting anti-dinitrotolamine monoclonal antibody and application thereof | |
| CN114752568B (en) | Furosemide monoclonal antibody, hybridoma cell strain and application | |
| CN106929479B (en) | Vitamin B2 monoclonal antibody hybridoma cell strain GZ-4 and application thereof | |
| CN113774030B (en) | Hybridoma cell strain secreting anti-picloram monoclonal antibody and application thereof | |
| CN112574957B (en) | Hybridoma cell strain secreting clomazone monoclonal antibody and application thereof | |
| CN113980909B (en) | Alpha-amanitin artificial antigen, monoclonal antibody, hybridoma cell strain and application | |
| CN115322969B (en) | Anti-dichlormid monoclonal antibody, monoclonal cell strain and application | |
| CN114774368B (en) | Hybridoma cell strain secreting anti-flumioxazin monoclonal antibody and application thereof | |
| CN114908059B (en) | Bispyribac-sodium monoclonal antibody hybridoma cell strain and application thereof | |
| CN111454912A (en) | Cyperazine monoclonal antibody hybridoma cell strain and application thereof | |
| CN115109757B (en) | Hybridoma cell strain secreting novobiocin monoclonal antibody and application thereof | |
| CN114292335B (en) | Hybridoma cell strain secreting TBHQ monoclonal antibody and application thereof | |
| CN113881638B (en) | De-himbine hapten, monoclonal antibody, hybridoma cell strain and application | |
| CN114774366B (en) | Hybridoma cell strain secreting flupirfuranone monoclonal antibody and application thereof | |
| CN114317450B (en) | Hybridoma cell strain secreting Flurobendiamide monoclonal antibody and application thereof | |
| CN114807051B (en) | Hybridoma cell strain of anti-dechloridone monoclonal antibody and application thereof | |
| CN113502272B (en) | Amaranth and carmine monoclonal antibody hybridoma cell strain and application thereof | |
| CN114958775B (en) | Rice blast amide artificial antigen, monoclonal antibody, hybridoma cell strain and application thereof | |
| CN114480295B (en) | Hybridoma cell strain secreting anti-butralin monoclonal antibody and application thereof |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |