CN114181911B - Hybridoma cell strain secreting spirolactone and metabolite monoclonal antibody thereof and application of hybridoma cell strain - Google Patents
Hybridoma cell strain secreting spirolactone and metabolite monoclonal antibody thereof and application of hybridoma cell strain Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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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
- G01N33/577—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
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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
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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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
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- 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
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- 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 spirolactone and a metabolite monoclonal antibody thereof, which is named as a monoclonal cell strain PRM and is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the year 2021, wherein the preservation address is the North Silu No.1, 3 of the Korean region of Beijing city, and the preservation number is CGMCC No.22341. The hybridoma cell strain can efficiently and stably secrete the spirolactone and the metabolite monoclonal antibody thereof, provides a powerful detection method and means for detecting the residual quantity of the spirolactone and the metabolite thereof in animal-derived foods and cosmetics, and has better sensitivity and specificity.
Description
Technical Field
The invention relates to the technical field of immunodetection, in particular to a hybridoma cell strain secreting spirolactone and a metabolite monoclonal antibody thereof and application thereof.
Background
Spironolactone, also known as Anshenshutong, is a mineralocorticoid antagonist developed by the company pyroxene, U.S. The spirolactone has a chemical structure similar to that of aldosterone, is an aldosterone receptor antagonist used earliest in clinic, can competitively bind with aldosterone receptor in cytoplasm, antagonize potassium and sodium expelling effect of aldosterone, promote water expelling, and reduce K + Is secreted by the host. Since this drug diuretic effect is related to the level of aldosterone in the body, it is a potassium-preserving diuretic. Clinically, the medicine is often combined with a cord diuretic to prevent hypokalemia in the treatment of hypertension. The spironolactone has low blood concentration and high metabolism speed in human body, and is metabolized into canrenone by liver after entering the human body. At present, diuretics are used as veterinary drugs to be added to feeds for treating bovine mastitis, or improving the protein proportion of poultry eggs, etc. However, some illegal molecules can use diuretics to cover up the use of other illegal drugs, and the purpose that the concentration of the illegal drugs in urine is reduced and discharged out of the body as soon as possible is achieved through the diuretics, so that the illegal drugs pass through drug detection and the like. Meanwhile, some illegal merchants add chemical medicines such as spironolactone into hair-growing cosmetics to obtain the effect of promoting hair growth in a short period, so that the long-term use of the hair-growing cosmetics by consumers under the condition of unknowing can lead to hormone-dependent dermatitis, and also can cause systemic side effects through skin absorption, so that the immune function is reduced, metabolic disorder is caused, and even cancer risks are caused. Both our cosmetic safety specifications (2015 edition) and European Union cosmetic regulations (EC) No.1223/2009 prescribe that such chemical drugs are forbidden to cosmetics.
The spirolactone and its metabolite content analysis method has the instrument methods such as liquid chromatography-mass spectrometry (LC-MS), and the detection methods have the defects of time consumption, complicated steps, incapability of carrying out on-site rapid detection, high cost and the like, so that the establishment of the rapid and simple spirolactone and its metabolite detection method has important significance. The enzyme-linked immunosorbent assay (ELISA) is a very efficient, sensitive and rapid detection method, is suitable for on-site rapid detection of a large number of samples, and provides a novel detection way for spironolactone and metabolites thereof.
Disclosure of Invention
In order to solve the technical problems, the invention provides a hybridoma cell strain secreting spirolactone and a monoclonal antibody of a metabolite thereof, a preparation method thereof, and detection of spirolactone and the metabolite thereof by using the strain and the monoclonal antibody produced by the strain.
The first object of the present invention is to provide a hybridoma cell strain secreting spirolactone and its metabolite monoclonal antibody, named monoclonal cell strain PRM, which is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) at the year 2021, month 05 and day 13, with a preservation address of Beijing, chaoyang, no.1, hospita 3, and a preservation number of CGMCC No.22341.
Further, the preparation method of the hybridoma cell strain comprises the following steps:
s1: preparing Cheng Fushi adjuvant from spirolactone and its metabolite immunogen, and injecting Freund's adjuvant into immunized animal; complete Freund's adjuvant is adopted for the first immunization, incomplete Freund's adjuvant is adopted for the multiple boosting immunization, spironolactone and metabolite immunogen thereof are adopted for the last sprint immunization;
the spirolactone and the metabolite immunogen thereof are prepared from a canrenone hapten, and the structure of the canrenone hapten is shown as a formula I;
s2: the immune animals subjected to the immunization process are subjected to blood sampling, the serum immune titer and the immune inhibition capacity of the immune animals are detected, and the immune animals with high content of spironolactone and metabolite antibodies thereof in serum are screened out;
s3: fusing and culturing spleen cells and myeloma cells of the immune animals obtained by screening in the step S2, detecting positive cell holes, determining the inhibition effect of the positive cell holes, and subcloning the positive cell holes with the best inhibition effect to obtain hybridoma cell strains secreting spironolactone and metabolite monoclonal antibodies thereof;
further, N-hydroxysuccinimide and 1-ethylcarbodiimide hydrochloride were used to activate spironolactone and its metabolite hapten.
Further, the interval between the first immunization and the boosting is 28-30 days, the interval between the boosting is 20-22 days, and the interval between the boosting and the sprint immunization is 18-21 days.
Further, the dose of the primary immunization is 95-105 mug/30 g of body weight, the dose of the boosting immunization is 45-55 mug/30 g of body weight, and the dose of the sprint immunization is 20-30 mug/30 g of body weight.
Further, in step S1, freund' S adjuvant is injected subcutaneously into the immunized animal via the back.
Further, in step S1, spironolactone and its metabolite immunogens are activated by canrenone hapten and then conjugated with bovine serum albumin.
Further, in step S3, cell fusion is performed 3 days after the termination of the sprint immunization.
Further, in step S3, the fused cells are cultured on RPMI-1640 medium.
The second object of the invention is to provide a spirolactone and its metabolite monoclonal antibody secreted by hybridoma cell strain with preservation number of CGMCC No.22341.
Further, paraffin oil is injected into the abdominal cavity of the immunized animal, then hybridoma cell strain with the preservation number of CGMCC No.22341 is injected into the abdominal cavity, ascites is collected after injection, and the ascites is purified, so that the spirolactone and the metabolite monoclonal antibody thereof are obtained.
The third purpose of the invention is to provide the application of the hybridoma cell strain or the spirolactone and the metabolite monoclonal antibody thereof in detecting spirolactone and the metabolite thereof, in particular to the application in the analysis and detection of spirolactone and the metabolite residue thereof in food, cosmetic or medicine safety monitoring, such as the preparation of an immune detection kit of spirolactone and the metabolite thereof and a colloidal gold test strip.
Further, metabolites include canrenone, testosterone, progesterone, baodarone, testosterone propionate, dehydroandrosterone, diflunisal, hydrocortisone, and the like.
A spirolactone and its metabolite detection kit comprising said hybridoma cell line and/or spirolactone and its metabolite monoclonal antibody.
Further, the spirolactone and metabolite thereof detection kit also comprises a spirolactone and metabolite coating antigen thereof, wherein the spirolactone and metabolite coating antigen thereof is obtained by coupling with chicken ovalbumin after activation of a spirolactone hapten, and the structure of the spirolactone hapten is shown as a formula II;
further, the preparation method of the spirolactone and the metabolite hapten thereof comprises the following steps:
(1) Dissolving spironolactone or canrenone and 2- (aminooxy) acetic acid (CMO) in DMF (N, N-dimethylformamide), heating in water bath at 50-80deg.C for 5-10 hr, and removing organic solvent to obtain yellow precipitate;
(2) Alternately washing with deionized water and 1M HCl, adding ethanol, and recrystallizing to obtain pale yellow or white needle-like crystals, and obtaining hapten Spi-COOH (spirolactone hapten) or Can-COOH (canrenone hapten) of spirolactone and its metabolite.
By means of the scheme, the invention has at least the following advantages:
the hybridoma cell strain provided by the invention can efficiently and stably secrete spirolactone and a metabolite monoclonal antibody thereof, and the monoclonal antibody has better specificity (the crossing rate of spirolactone and a metabolite analogue thereof is less than 2.2%) and detection sensitivity (spirolactone IC) 50 The value was 0.23ng/mL, canrenone IC 50 The value is 0.18 ng-mL), can realize the detection of the residual quantity of spirolactone and metabolites thereof in animal-derived foods, cosmetics or medicines, and has practical application value.
The foregoing description is only an overview of the present invention, and is presented in terms of preferred embodiments of the present invention and the following detailed description of the invention in conjunction with the accompanying drawings.
Preservation of biological materials
The monoclonal cell strain PRM is preserved in China general microbiological culture collection center (CGMCC) No.22341 at the year of 2021, month 05 and 13, and the preservation address is North Chen Xiyu No.1 and 3 in the Chaoyang area of Beijing city.
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 that are illustrated in the appended drawings.
FIG. 1 is a standard curve of the inhibition of spironolactone by a monoclonal antibody of the present invention and its metabolite;
FIG. 2 is a standard curve of inhibition of canrenone by the spirolactone and its metabolite monoclonal antibodies of the present 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.
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 B12.005, sodium bicarbonate 2000.
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 of pH7.4 was added with 0.5. 0.5 mL of Tween-20;
PBST: PBS containing 0.05% Tween-20;
antibody dilution: a wash buffer containing 0.1% gelatin;
TMB color development liquid: and (3) solution A: na (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 1: 5, mixing to obtain TMB color development liquid, and mixing immediately.
The detection method involved in the following examples is as follows:
the method for detecting the inhibition rate of spironolactone and the metabolite thereof 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 0.03,0.1,0.3 and 1 μg/mL with Carbonate Buffer (CBS) and the antibody diluted to 0.03,0.1,0.3 and 1 μg/mL with antibody dilution. After selecting the best working point, the spironolactone and the metabolite standard thereof are diluted to the equal concentrations of 0,0.04,0.12,0.37, 1.11,3.33, 10 and 30ng/mL, and finally the initial Pro 8.5 is used for drawing according to the IC-ELISA operation step to obtain the spironolactoneLactone and metabolite standard inhibition curve, IC was calculated 50 。
EXAMPLE 1 Synthesis of spirolactone and its metabolite hapten
1g of spironolactone (or canrenone) and 3g of CMO were dissolved in 10mL of DMF and heated in a water bath at 60℃for 8h. The organic solvent was removed by rotary evaporation under reduced pressure to give a yellow precipitate. Washing with deionized water and 1M HCl for three times, adding ethanol, and recrystallizing to obtain pale yellow or white needle-like crystals, i.e. hapten Spi-COOH (or Can-COOH) of spironolactone and its metabolite.
The reaction equation is shown below:
EXAMPLE 2 Synthesis of spirolactone and its metabolite complete antigen
(1) Preparation of the immunogen spirolactone and its metabolite-BSA: 3.4mg of prepared Can-COOH is weighed and dissolved in 200 mu L of DMF, 4.6mg of N-hydroxysuccinimide and 7.7mg of 1-ethylcarbodiimide hydrochloride are added in sequence under stirring, and the mixture obtained is called A solution after reaction for 4 hours at room temperature; then 10mg of bovine serum albumin BSA was weighed and dissolved in 2mL of carbonate buffer, designated as solution B; slowly dripping the solution A into the solution B, reacting for 12 hours at room temperature under stirring, dialyzing for 3d by using 0.01mol/L phosphate buffer PBS, and obtaining the conjugate spironolactone and a metabolite thereof-BSA, and freezing at-20 ℃ for later use;
(2) Preparation of coating raw spirolactone and metabolite-OVA thereof: 1.7mg of the Spi-COOH prepared was weighed and dissolved in 200. Mu.L of DMF, 2.3mg of N-hydroxysuccinimide and 3.4mg of 1-ethylcarbodiimide hydrochloride were added in sequence under stirring, and the mixture was reacted at room temperature for 4 hours, the obtained mixture was referred to as solution A; then 10mg of chicken ovalbumin OVA is weighed and dissolved in 2mL of carbonate buffer solution, called as B solution; slowly dripping the solution A into the solution B, reacting for 12 hours at room temperature under stirring, dialyzing for 3d by using 0.01mol/L phosphate buffer PBS, and obtaining the conjugate spironolactone and a metabolite thereof, namely OVA, and freezing at-20 ℃ for later use.
EXAMPLE 3 preparation of hybridoma cell lines secreting spironolactone and its metabolite monoclonal antibodies
1. Immunization of mice: healthy BALB/c mice of 6-8 weeks of age were selected for immunization. Mixing spironolactone and its metabolite complete antigen with equivalent Freund's adjuvant, emulsifying, and immunizing BALB/c mice respectively by back subcutaneous injection. The first immunization was with complete Freund's adjuvant, followed by incomplete Freund's adjuvant. The interval between the first immunization and the second boost was 28 days, and the interval between the multiple boosts was 21 days. Blood was collected 7 days after the third immunization (5 μl of mouse tail-cut blood collection+995μl of antibody diluent=antisera), serum titers and inhibition were determined using ic-ELISA, mice with high titers were selected for sprint immunization 21 days after the fifth immunization, i.p. injection, required half the dosage of the wash-out and no adjuvant.
2. Cell fusion: three days after sprint immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 4000) method, as follows:
(1) Taking eyeball and blood, killing a mouse by a cervical dislocation method, immediately putting the mouse into 75% alcohol for disinfection, soaking for about 5min, taking out spleen of the mouse by aseptic operation, moderately grinding the spleen by a rubber head of a syringe, obtaining spleen cell suspension by a 200-mesh cell screen, collecting, centrifuging (1200 rpm,8 min), washing the spleen cells for three times by using an RPMI-1640 culture medium, and diluting the spleen cells to a certain volume and counting for later use after the last centrifugation;
(2) Collecting murine myeloma SP2/0 cells: SP2/0 tumor cells were cultured in a 5% CO2 incubator with 10% FBS (fetal bovine serum) RPMI-1640 medium 7-10 days prior to fusion. The number of SP2/0 tumor cells before fusion reaches 1 to 4 multiplied by 107, so that the SP2/0 tumor cells before fusion are ensured to be in logarithmic growth phase. During fusion, collecting tumor cells, suspending in RPMI-1640 basic culture solution, and performing cell count;
(3) The fusion process was 7min. 1min, 1mL of PEG 1500 was added dropwise to the cells from slow to fast; 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 mixture was incubated at 37℃for 5min. Centrifugation (800 rpm,8 min), discarding supernatant, re-suspending in RPMI-1640 screening medium containing 20% fetal bovine serum and 2% 50 XHAT, adding 200. Mu.L/well to 96 well cell plates, and culturing in a 5% CO2 incubator at 37 ℃.
3. 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 select spironolactone and metabolites thereof as standard substances, and to measure the inhibition effect of positive cells by using ic-ELISA. Cell holes with better inhibition on spirolactone and metabolite standard thereof are selected, subcloning is carried out by adopting a limiting dilution method, and detection is carried out by adopting the same method. Cell lines were obtained by repeating the procedure three times.
EXAMPLE 4 preparation and identification of spirolactone and its metabolite monoclonal antibodies
Taking 8-10 week old BALB/c mice, and injecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; after 7 days, 1X 106 hybridoma cells were intraperitoneally injected into each mouse, and ascites was collected from the seventh day and purified by the octanoic acid-ammonium sulfate method. Under the condition of meta-acid, the n-octanoic acid can precipitate other hetero proteins except IgG immunoglobulin in ascites, and then the mixture is centrifuged and the precipitate is discarded; precipitating the monoclonal antibody of the IgG type by using an ammonium sulfate solution with equivalent saturation, centrifuging, discarding the supernatant, dissolving by using a 0.01M PBS solution (pH 7.4), dialyzing for desalting, and finally obtaining the purified monoclonal antibody, and storing at-20 ℃.
Sensitivity detection: determination of monoclonal antibody versus spirolactone IC using indirect competition ELISA method 50 The value is 0.23ng/mL, and canrenone IC 50 The value was 0.18ng/mL.
And (3) specificity detection: cross-over = (IC of spirolactone and its metabolite) 50 IC of analog 50 ) X 100%, IC of spirolactone and its metabolite analogue 50 The values and crossover rates are shown in the following table.
TABLE 1 IC of spirolactone and its metabolite analogs 50 Value of
EXAMPLE 5 application of spirolactone and its metabolite monoclonal antibody
The monoclonal antibody prepared from the hybridoma cell strain through in-vivo ascites is applied to an additive recovery test of spironolactone and metabolites thereof, and the specific steps are as follows:
(1) Coating: the coated plaalolide and its metabolite-OVA were diluted in a 1. Mu.g/mL ratio starting with 0.05M carbonate buffer at pH 9.6, 100. Mu.L/well, and reacted at 37℃for 2h.
(2) Washing: the plate solution was poured off and washed 3 times with wash solution for 3min each.
(3) 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.
(4) Sample adding: the antiserum (after tail breaking and blood sampling of the mice, the antiserum is diluted by corresponding times by antibody diluent) is diluted by a ratio of 1:1000, and is added into the coating holes of each dilution, 100 mu L/hole and reacted for 30min at 37 ℃; after extensive washing, HRP-goat anti-mouse IgG diluted 1:3000 was added, 100. Mu.L/well, and reacted at 37℃for 30 minutes.
(5) Color development: and taking out the ELISA plate, fully washing, adding 100 mu L of TMB color developing solution into each hole, and carrying out light-shielding reaction for 15min at 37 ℃.
(6) 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.
Determination of monoclonal antibody to spirolactone IC by IC-ELISA 50 The value is 0.23ng/mL, and canrenone IC 50 The value is 0.18ng/mL, which shows that the kit has good sensitivity to spironolactone and metabolites thereof, and can be used for immunoassay detection of spironolactone and metabolites thereof.
The standard curves of inhibition of spironolactone and its metabolites by the monoclonal antibodies to spironolactone and its metabolites are shown in fig. 1 (spironolactone) and fig. 2 (canrenone). The antibody has better sensitivity to spironolactone and metabolites thereof, can be used for immunoassay detection of spironolactone and metabolites thereof, and establishes an immunological detection method of spironolactone and metabolites thereof.
Comparative example 1
The immunogen is replaced by Spi-COOH-BSA, the coating antigen is selected from Spi-COOH-OVA and Can-COOH-OVA, and the inhibition effect of mouse serum on spironolactone and canrenone is verified by the rest steps as in the example, and the results are shown in tables 2 and 3.
TABLE 2 inhibition of spironolactone and canrenone when the coating antigen is Spi-COOH-OVA
Note that: "-" is reading exceeding detection range of enzyme label instrument, which indicates high serum potency.
TABLE 3 inhibition of spironolactone and canrenone when the coating antigen is Can-COOH-OVA
Note that: "-" is reading exceeding detection range of enzyme label instrument, which indicates high serum potency.
Comparative example 2
The immunogen still selected Can-COOH-BSA, the coating antigen selected Spi-COOH-OVA and Can-COOH-OVA, and the other steps were the same as in the examples, and the inhibitory effects of mouse serum on spironolactone and canrenone were verified, and the results are shown in tables 4 and 5.
TABLE 4 inhibition of spironolactone and canrenone when the coating antigen is Can-COOH-OVA
Note that: "-" is reading exceeding detection range of enzyme label instrument, which indicates high serum potency.
TABLE 5 inhibition of spironolactone and canrenone when the coating antigen is Spi-COOH-OVA
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Note that: "-" is reading exceeding detection range of enzyme label instrument, which indicates high serum potency.
As Can be seen from tables 2-5, when the immunogen is Spi-COOH-BSA and the coating is Spi-COOH-OVA and Can-COOH-OVA, the mouse serum has no better inhibition on both drugs: when the immunogen is Can-COOH-BSA and the coating is Can-COOH-OVA, only spirolactone is inhibited, and when the coating is Spi-COOH-OVA, both medicines are inhibited.
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 (7)
1. A hybridoma cell line secreting spirolactone and its metabolite monoclonal antibodies, characterized in that: the hybridoma cell strain is named as a monoclonal cell strain PRM and is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on the 13 th day of 2021, wherein the preservation address is the North Chen Xili No.1, 3 in the Korean region of Beijing, and the preservation number is CGMCC No.22341; the metabolite is canrenone.
2. A spirolactone and its metabolite monoclonal antibody, characterized in that: the spirolactone and the metabolite monoclonal antibody thereof are secreted by the hybridoma cell strain of claim 1; the metabolite is canrenone.
3. Use of the hybridoma cell line of claim 1 or the monoclonal antibody of claim 2 for detecting spironolactone or canrenone.
4. A spirolactone and metabolite detection kit thereof is characterized in that: the spironolactone and metabolite detection kit comprises the spironolactone and metabolite monoclonal antibody thereof according to claim 2.
5. The spironolactone and metabolite detection kit of claim 4, wherein: the spirolactone and metabolite detection kit also comprises spirolactone and metabolite coating antigen thereof.
6. The spironolactone and metabolite detection kit of claim 5, wherein: the spirolactone and the metabolite coating antigen thereof are obtained by coupling chicken ovalbumin after activation of spirolactone hapten.
7. The spironolactone and metabolite detection kit of claim 6, wherein: the structure of the spirolactone hapten is shown as a formula II:
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| CN113621583A (en) * | 2021-09-17 | 2021-11-09 | 江南大学 | Hybridoma cell strain secreting dimethomorph monoclonal antibody and application thereof |
| CN113684187A (en) * | 2021-09-22 | 2021-11-23 | 江南大学 | Hybridoma cell strain secreting monoclonal antibody to fluazifop-butyl as well as preparation method and application of hybridoma cell strain |
| CN113717949A (en) * | 2021-09-22 | 2021-11-30 | 江南大学 | Hybridoma cell strain capable of secreting ketoconazole monoclonal antibody and application thereof |
| CN113736744A (en) * | 2021-10-14 | 2021-12-03 | 江南大学 | Digitalis glycosides monoclonal antibody hybridoma cell strain and application thereof |
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| CN113621583A (en) * | 2021-09-17 | 2021-11-09 | 江南大学 | Hybridoma cell strain secreting dimethomorph monoclonal antibody and application thereof |
| CN113684187A (en) * | 2021-09-22 | 2021-11-23 | 江南大学 | Hybridoma cell strain secreting monoclonal antibody to fluazifop-butyl as well as preparation method and application of hybridoma cell strain |
| CN113717949A (en) * | 2021-09-22 | 2021-11-30 | 江南大学 | Hybridoma cell strain capable of secreting ketoconazole monoclonal antibody and application thereof |
| CN113736744A (en) * | 2021-10-14 | 2021-12-03 | 江南大学 | Digitalis glycosides monoclonal antibody hybridoma cell strain and application thereof |
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