CN113736744B - Digitoxin monoclonal antibody hybridoma cell strain and application thereof - Google Patents
Digitoxin monoclonal antibody hybridoma cell strain and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of food safety immunodetection, and in particular relates to a digitoxin monoclonal antibody hybridoma cell strain FJN and application thereof, wherein the cell strain is preserved in China general microbiological culture collection center (CGMCC) with the address of China national academy of sciences of China No. 3 of the national academy of sciences of North Chen West Lu No. 1 in the Korean region of Beijing, and the preservation date is 2021, 05 and 13 days, and the preservation number is CGMCC No.22325. The monoclonal antibody secreted by the digitoxin monoclonal antibody hybridoma cell strain provided by the invention has better specificity and detection sensitivity (IC) to digitoxin 50 The value is 0.51 ng/mL), and provides an immunological method for detecting the digitoxin content in clinical blood samples. The digitoxin monoclonal antibody hybridoma cell strain and the monoclonal antibody secreted by the same can be prepared into a kit for detecting digitoxin, and have practical application value.
Description
Technical Field
The invention belongs to the field of food safety immunodetection, and particularly relates to a digitoxin monoclonal antibody hybridoma cell strain FJN and application thereof.
Background
Digitoxin (Digitoxin), also known as Digitoxin Ji Tuoxin, digitoxin or Di Ji Tuoxin, is an oral cardiac glycoside preparation, which is a white crystalline powder, odorless, slightly soluble in chloroform, slightly soluble in ethanol, diethyl ether, and insoluble in water. Cardiac glycoside medicine is used clinically in treating cardiac failure and arrhythmia, and belongs to the field of Na + /K + Atpase inhibitors. Digitoxin and Na + /K + The ATPase is reversibly combined on the cell membrane, inhibits active transport of sodium ions and potassium ions, so that the concentration of sodium ions in the cell is increased, which in turn promotes exchange of sodium ions and calcium ions, and improves the concentration of calcium ions in the cell. Calcium ions play an important role in cardiac excitation and contraction, which is the basis of the cardiotonic effect of digitoxin. Digitalis is usually indicated for chronic cardiac insufficiency, especially renal insufficiency, because it is slow and durable, and is not used instead of digoxin. Digitoxin also has many disadvantages such as easy accumulation in human body, small difference between therapeutic and toxic amounts, and large individual differences in patient sensitivity and tolerance. The doctor should adjust the dosage of digitoxin in time in combination with the measurement of the blood concentration of digitoxin and clinical symptoms of the patient. Thus, there is a need to establish a rapid, efficient method of digitoxin monitoring.
The digitoxin detection method is usually an instrument method such as High Performance Liquid Chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS), and has 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 digitoxin 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 digitoxin detection.
Disclosure of Invention
The invention aims to provide a hybridoma cell strain of a digitoxin monoclonal antibody and application thereof, and the antibody prepared by the cell strain has better specificity and detection sensitivity to the digitoxin and can be used for establishing an immunological detection method of the digitoxin.
According to the technical scheme of the invention, the digitoxin monoclonal antibody hybridoma cell strain FJN is preserved in China General Microbiological Culture Center (CGMCC) with the address of China academy of sciences of China, including national institute of microbiology, national institute of sciences, no. 3, national center for sciences, no. 1, north Chen, and the area of Korea, beijing, and the preservation date 2021, no. 05, and the preservation number is CGMCC No.22325.
In a second aspect, the present invention provides a method for preparing the digoxigenin monoclonal antibody hybridoma cell strain FJN, comprising the steps of,
s1: coupling digitoxin with carrier protein to obtain immunogen;
s2: mixing and emulsifying the immunogen and Freund's adjuvant, performing subcutaneous immunization on mice, and screening the immunized mice with high digitoxin antibody content in serum;
s3: and (2) fusing spleen cells and myeloma cells of the mice screened in the step (S2) to obtain the digitoxin monoclonal antibody hybridoma cell strain FJN.
Further, the carrier protein is Bovine Serum Albumin (BSA) or Keyhole Limpet Hemocyanin (KLH), and the immunogens obtained by the carrier protein are digitoxin-BSA and digitoxin-KLH respectively.
Further, the specific operation of the step S1 is as follows,
SS1: dissolving digitoxin in a solvent, sequentially adding sodium periodate and ethylene glycol, and stirring to obtain a mixed solution;
SS2: adding the mixed solution into carrier protein, and regulating the pH value to 9-10;
SS3: adding sodium borohydride, and reacting to obtain the immunogen.
Further, in the step SS1, the molar ratio of digitoxin to sodium periodate is 1:3-4, the molar ratio of digitoxin to glycol is 1:2-3.
Further, the solvent is dimethyl sulfoxide (DMSO).
Further, when the carrier protein is BSA, the molar ratio of digitoxin to carrier protein is 30-90:1, a step of; when the carrier protein is KLH, the molar ratio of digitoxin to carrier egg is 3000-9000:1.
further, in the step SS2, K is added 2 CO 3 Solution or Na 2 CO 3 The pH of the solution is adjusted to 8-10.
Further, in the step SS3, sodium borohydride solution is added for reduction reaction, formic acid or hydrochloric acid is added for adjusting the pH value to 6-7, standing is carried out, and ammonia water or NaOH solution is added for adjusting the pH value to 8-9, so that the immunogen is prepared.
In step S2, the first immunization is performed by mixing and emulsifying the immunogen and the complete freund 'S adjuvant, the multiple boosting is performed by mixing and emulsifying the immunogen and the incomplete freund' S adjuvant, and the final immunization is performed by intraperitoneal injection after diluting the immunogen with normal saline.
Specifically, the first immunization dose is 100 mug/dose; multiple booster doses were 50 μg/dose; one month is separated from the first immunization and the second immunization, and 21 days is separated from the multiple immunization; the interval between the sprint immunity and the last boost immunity is 18-21 days.
In the step S3, the mouse spleen cells screened in the step S2 are fused with the mouse myeloma cells by a polyethylene glycol (PEG 4000) method, the positive cell holes are detected by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method through a selective medium (HAT medium) culture, the inhibition effect of the positive cell holes is further determined by the ic-ELISA method, the positive cell holes with the best inhibition effect are subcloned for three times by a limiting dilution method, and the digitoxin monoclonal antibody hybridoma cell strains are finally obtained through screening.
In a third aspect, the present invention provides a digoxigenin monoclonal antibody, which is secreted by the digoxigenin monoclonal antibody hybridoma cell strain FJN with a preservation number of CGMCC No.22325.
In a fourth aspect, the invention provides the use of a digitoxin monoclonal antibody as described above in a digitoxin assay.
In particular, it can be used for monitoring digitoxin concentration in clinical blood samples.
The invention also provides a kit for detecting digitoxin, which comprises the digitoxin monoclonal antibody; and a kit for detecting digitoxin, comprising the digitoxin monoclonal antibody.
Compared with the prior art, the technical scheme of the invention has the following advantages: the monoclonal antibody secreted by the digitoxin monoclonal antibody hybridoma cell strain provided by the invention has better specificity and detection sensitivity (IC) to digitoxin 50 The value is 0.51 ng/mL), and provides an immunological method for detecting the digitoxin content in clinical blood samples. The digitoxin monoclonal antibody hybridoma cell strain and the monoclonal antibody secreted by the same can be prepared into a kit for detecting digitoxin, and have practical application value.
Preservation of biological materials: the digitoxin monoclonal antibody hybridoma cell strain FJN is preserved in China General Microbiological Culture Center (CGMCC) with the address of China academy of sciences of China, national academy of sciences of China, no. 1, north Chengxi, and the Korean area, and classified and named as a monoclonal cell strain, wherein the preservation date is 2021, 05, 13 days, and the preservation number is CGMCC No.22325.
Drawings
FIG. 1 is a standard curve of digitoxin inhibition by digitoxin monoclonal antibodies.
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 solutions in the following examples were formulated as follows:
carbonate Buffer (CBS): weighing Na 2 CO 3 1.59 g，NaHCO 3 2.93 g, respectively dissolving in a small amount of double distilled water, mixing, adding the double distilled water to about 800mL, mixing uniformly, adjusting the pH value to 9.6, adding the double distilled water to 1000mL, and storing at 4 ℃ for later use.
Phosphate Buffer (PBS): 8.0g 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 0.5mL 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. And mixing the solution B according to the volume ratio of 1:5 to obtain TMB color development solution, wherein the solution B is mixed immediately before use.
Example 1 preparation of hybridoma cell line FJN
(1) Preparation of the immunogen digitoxin-BSA:
21.4mg digitoxin is weighed and dissolved in 1mL dimethyl sulfoxide (DMSO), and 0.1M sodium periodate (NaIO) is added dropwise 4 ) 1mL of solution, and stirring for 1h at room temperature; 60 mu L of 1M glycol is added and stirring is continued for 5min; the above mixed reaction solution was added dropwise to 28mg/mL of Bovine Serum Albumin (BSA) solution, followed by 5% potassium carbonate (K 2 CO 3 ) Adjusting the pH to 9, and continuously stirring for 1h until the pH is stable; 15mg of sodium borohydride (NaBH) was weighed 4 ) Dissolving in water, and adding into the reaction solution to react for 16h; adding 1M formic acid, adjusting pH to 65, standing at room temperature for 1h; adding ammonia (NH) 4 OH), adjusting pH to 8.5; dialyzing the reaction solution for 24 hours; after dialysis, 0.1M hydrochloric acid (HCl) was adjusted to pH 5, the precipitate was allowed to stand at room temperature for 1h, and at 4℃for 3h; centrifuge at 12000rpm for 8min, precipitate with sodium bicarbonate (NaHCO 3 ) Dissolving, and separating complete antigen and unconjugated small molecule hapten by dialysis to obtain the immunogen digitoxin-BSA.
(2) Preparation of coated digoxigenin-OVA:
21.4mg digitoxin is weighed and dissolved in 1mL dimethyl sulfoxide (DMSO), and 0.1M sodium periodate (NaIO) is added dropwise 4 ) 1mL of solution, and stirring for 1h at room temperature; 60 mu L of 1M glycol is added and stirring is continued for 5min; the above mixed reaction solution was added dropwise to 28mg/mL chicken Ovalbumin (OVA) solution, followed by 5% potassium carbonate (K 2 CO 3 ) Adjusting the pH to 9, and continuously stirring for 1h until the pH is stable; 15mg of sodium borohydride (NaBH) was weighed 4 ) Dissolving in water, and adding into the reaction solution to react for 16h; adding 1M formic acid, adjusting the pH to 6.5, and standing at room temperature for 1h; adding ammonia (NH) 4 OH), adjusting pH to 8.5; dialyzing the reaction solution for 24 hours; after dialysis, 0.1M hydrochloric acid (HCl) was adjusted to pH 5, the precipitate was allowed to stand at room temperature for 1h, and at 4℃for 3h; centrifuge at 12000rpm for 8min, precipitate with sodium bicarbonate (NaHCO 3 ) Dissolving, and separating complete antigen and unconjugated small molecule hapten by dialysis to obtain the immunogen digitoxin-OVA.
(3) Animal immunization: healthy 6-8 week old BALB/c mice were selected for immunization. After mixing and emulsifying digitoxin complete antigen (immunogen digitoxin-BSA) with an equal volume of Freund's adjuvant, BALB/c mice were immunized by back subcutaneous injection, respectively. Complete Freund's adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used for the multiple boosting. One month is separated from the first immunization and the second immunization, 21 days is separated from the multiple immunization, and the final immunization is performed by intraperitoneal injection (without adjuvant) after the immunogen is diluted by normal saline for the last time, and 18-21 days is separated from the final immunization. The ic-ELISA was used to determine mouse serum titers and inhibition, and mice with high titers and good inhibition were selected.
(4) Cell fusion: three days after sprint immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 4000) method, as follows:
a. 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;
b. collecting SP2/0 cells: SP2/0 tumor cells were cultured in 10% FBS (fetal bovine serum) RPMI-1640 medium at 5% CO 7-10 days prior to fusion 2 In an incubator. The number of SP2/0 tumor cells is 1-4×10 before fusion 7 Ensuring SP2/0 tumor cells to be in 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 into the cells from slow to fast in a dropwise manner; 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, gently tapping cells, resuspending in RPMI-1640 medium containing 20% fetal bovine serum, 2% 50 XHAT, 200. Mu.L/well onto 96 well cell plates, and incubating at 37℃with 5% CO 2 Culturing in an incubator.
(5) 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 digitoxin as a standard substance, and to measure the inhibition effect of positive cells by using ic-ELISA. Cell holes with better inhibition on digitoxin standard substances are selected, subcloning is carried out by adopting a limiting dilution method, and detection is carried out by adopting the same method. Cell line FJN was obtained by repeating three times.
(6) Preparation and identification 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. 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 ℃.
EXAMPLE 2 IC of digoxigenin monoclonal antibody 50 Is (are) determined by
(1) Coating: the coated digoxigenin-OVA was diluted in a 1. Mu.g/mL ratio using 0.05M pH 9.6 carbonate buffer, 100. Mu.L/well, and reacted at 37℃for 2 hours.
(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: mu.L of stop solution was added to each well to terminate the reaction, and the OD (450 nm) value of each well was measured with a microplate reader.
IC for determining monoclonal antibody digitoxin by IC-ELISA 50 The sensitivity to digitoxin is 0.51ng/mL, which shows that the kit has good sensitivity to digitoxin and can be used for the immunoassay detection of the digitoxin.
The cross-reactions of digoxigenin monoclonal antibodies to digoxigenin, digoxin, acetyldigoxigenin, and ouabain were measured separately and the results are shown in table 1:
TABLE 1 specificity of monoclonal antibody 4B12
Note that: cross-reactivity (CR) =digitoxin IC 50 IC of structural analogue 50 *100％。
The results show that the digitoxin monoclonal antibody has extremely low cross reaction rate on digoxin, acetyldigitoxin and ouabain, so that the antibody prepared by the invention has better specificity on the digitoxin.
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 (5)
1. A digitoxin monoclonal antibody hybridoma cell strain FJN is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.22325, and is preserved on 2021, 05 and 13 days by the institute of microorganisms, national academy of sciences of China, no. 3, north Chen West Lu No. 1, the area of Beijing, and the preservation number of the strain is CGMCC No.22325.
2. A digoxigenin monoclonal antibody, which is secreted by the digoxigenin monoclonal antibody hybridoma cell line FJN of the collection number CGMCC No.22325 of claim 1.
3. Use of a digoxigenin monoclonal antibody according to claim 2, in the preparation of a digoxigenin detection reagent.
4. A composition for detecting digoxigenin, comprising the digoxigenin monoclonal antibody of claim 2.
5. A kit for detecting digoxigenin, comprising the digoxigenin monoclonal antibody of claim 2.
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| CN114181911B (en) * | 2021-12-28 | 2023-08-04 | 江南大学 | Hybridoma cell strain secreting spirolactone and metabolite monoclonal antibody thereof and application of hybridoma cell strain |
| CN114292335B (en) * | 2021-12-31 | 2023-06-30 | 江南大学 | Hybridoma cell strain secreting TBHQ monoclonal antibody and application thereof |
| CN116396392B (en) * | 2023-01-17 | 2023-10-27 | 珠海重链生物科技有限公司 | Antibody specific to digoxigenin and related application thereof |
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| US4469797A (en) * | 1982-09-23 | 1984-09-04 | Miles Laboratories, Inc. | Digoxigenin immunogens, antibodies, labeled conjugates, and related derivatives |
| US4742159A (en) * | 1983-11-26 | 1988-05-03 | Boehringer Mannheim Gmbh | Digitalis antibodies, process for the preparation thereof and the use thereof for the therapy of digitalis intoxications |
| US5656434A (en) * | 1990-12-28 | 1997-08-12 | Suntory Limited | Monoclonal antibody against cardiac glycoside and utilization thereof |
| CN103439485A (en) * | 2013-08-22 | 2013-12-11 | 天津市康婷生物工程有限公司 | Kit for efficiently detecting endogenous digitalis-like factor content and application method thereof |
| CN112285038A (en) * | 2019-01-09 | 2021-01-29 | 北京九强生物技术股份有限公司 | 6-phosphoglucose dehydrogenase mutant and application thereof in preparing digitoxin detection reagent |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4469797A (en) * | 1982-09-23 | 1984-09-04 | Miles Laboratories, Inc. | Digoxigenin immunogens, antibodies, labeled conjugates, and related derivatives |
| US4742159A (en) * | 1983-11-26 | 1988-05-03 | Boehringer Mannheim Gmbh | Digitalis antibodies, process for the preparation thereof and the use thereof for the therapy of digitalis intoxications |
| US5656434A (en) * | 1990-12-28 | 1997-08-12 | Suntory Limited | Monoclonal antibody against cardiac glycoside and utilization thereof |
| CN103439485A (en) * | 2013-08-22 | 2013-12-11 | 天津市康婷生物工程有限公司 | Kit for efficiently detecting endogenous digitalis-like factor content and application method thereof |
| CN112285038A (en) * | 2019-01-09 | 2021-01-29 | 北京九强生物技术股份有限公司 | 6-phosphoglucose dehydrogenase mutant and application thereof in preparing digitoxin detection reagent |
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