CN113736744A

CN113736744A - Digitalis glycosides monoclonal antibody hybridoma cell strain and application thereof

Info

Publication number: CN113736744A
Application number: CN202111199470.1A
Authority: CN
Inventors: 胥传来; 姜晓倩; 匡华; 徐丽广; 孙茂忠; 刘丽强; 吴晓玲; 宋珊珊; 胡拥明; 郝昌龙; 马伟; 吴爱红; 高巍
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-10-14
Filing date: 2021-10-14
Publication date: 2021-12-03
Anticipated expiration: 2041-10-14
Also published as: CN113736744B

Abstract

The invention belongs to the field of food safety immunodetection, and particularly relates to a digoxigenin monoclonal antibody hybridoma cell strain FJN and application thereof, wherein the cell strain is preserved in China general microbiological culture Collection center (CGMCC), the microbial research institute of China academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, the preservation date is 2021 year, 05 month and 13 days, and the preservation number is CGMCC No. 22325. The monoclonal antibody secreted by the digoxigenin monoclonal antibody hybridoma cell strain has good specificity and detection sensitivity (IC) on digoxigenin₅₀A value of 0.51ng/mL), provides an immunological method for detecting the digoxigenin content in a blood sample clinically. The digoxigenin monoclonal antibody hybridoma cell strain and the monoclonal antibody secreted by the same can be prepared into a kit for detecting digoxigenin, and have practical application values.

Description

Digitalis glycosides monoclonal antibody hybridoma cell strain and application thereof

Technical Field

The invention belongs to the field of food safety immunodetection, and particularly relates to a digoxigenin monoclonal antibody hybridoma cell strain FJN and application thereof.

Background

Digitoxin (Digitoxin), also known as Digitoxin, Digitoxin or Digitoxin, is an oral cardiac glycoside preparation, which is white crystalline powder, odorless, slightly soluble in chloroform, slightly soluble in ethanol and ether, and insoluble in water. Cardiac glycoside medicine is mainly used for treating heart failure and arrhythmia in clinic, and belongs to Na⁺/K⁺An ATPase inhibitor. Digitalis glycosides and Na⁺/K⁺The ATP enzyme is reversibly combined on cell membranes to inhibit active transport of sodium ions and potassium ions, so that the concentration of sodium ions in cells is increased, and the exchange of the sodium ions and calcium ions is promoted, and the concentration of calcium ions in the cells is increased. Calcium ions play an important role in cardiac excitation and contraction, which is the basis for digitoxin to play a cardiotonic role. Because the digitoxin has slow and lasting effect, the medicine is generally suitable for patients with chronic cardiac insufficiency, and particularly, digitoxin instead of digoxin is selected for patients with renal insufficiency. Digitoxin also has a number of disadvantages, such as a tendency to accumulate in humans, a small difference between therapeutic and toxic amounts, and a large individual variation in patient sensitivity and tolerance. The doctor should adjust the dosage of digitoxin in time by combining the blood concentration measurement and clinical symptoms of the digitoxin of the patient. Therefore, there is a need to establish a rapid and efficient method for monitoring digitoxin.

The digitoxin detection method is generally an instrument method such as a High Performance Liquid Chromatography (HPLC), a liquid chromatography-mass spectrometry (LC-MS) and the like, and has the defects of time consumption, complicated steps, incapability of performing on-site rapid detection, high cost and the like, so that the establishment of the rapid, simple and convenient digitoxin detection method has important significance. An enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive and rapid detection method, is suitable for the field rapid detection of a large number of samples, and provides a new detection way for digitoxin detection.

Disclosure of Invention

The invention aims to provide a hybridoma cell strain of a digoxigenin monoclonal antibody and application thereof, and the antibody prepared by the cell strain has better specificity and detection sensitivity on digoxigenin and can be used for establishing an immunological detection method of the digoxigenin.

According to the technical scheme of the invention, the digitoxin monoclonal antibody hybridoma cell strain FJN is preserved in China general microbiological culture collection center (CGMCC) of China Committee for culture Collection of microorganisms, and is prepared by the microbiological research institute of China academy of sciences, No. 3 of West Lu 1 Hospital, North Cheng, Chaoyang, the Beijing, with the preservation date of 2021 year, 05 month and 13 days and the preservation number of CGMCC No. 22325.

The second aspect of the invention provides a method for preparing the digoxigenin monoclonal antibody hybridoma cell strain FJN, which comprises the following steps,

s1: coupling digitoxin with carrier protein to obtain immunogen;

s2: mixing and emulsifying the immunogen and Freund's adjuvant, performing subcutaneous immunization on a mouse, and screening the immunized mouse with high digitoxin antibody content in serum;

s3: fusing spleen cells and myeloma cells of the mouse screened in the step S2 to obtain the digoxigenin monoclonal antibody hybridoma cell strain FJN.

Further, the immunogen obtained by the carrier protein is Bovine Serum Albumin (BSA) or Keyhole Limpet Hemocyanin (KLH) is digoxigenin-BSA and digoxigenin-KLH respectively.

Further, the specific operation of step S1 is as follows,

SS 1: dissolving digitoxin in a solvent, sequentially adding sodium periodate and ethylene glycol, and stirring to obtain a mixed solution;

SS 2: adding the mixed solution into carrier protein, and adjusting the pH to 9-10;

SS 3: and adding sodium borohydride, and reacting to obtain the immunogen.

Further, in 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; when the carrier protein is KLH, the molar ratio of the digoxigenin to the carrier protein is 3000-9000: 1.

further, in the step SS2, by adding K₂CO₃Solutions or Na₂CO₃The pH of the solution is adjusted to 8-10.

Further, in the step SS3, adding a sodium borohydride solution for reduction reaction, adding formic acid or hydrochloric acid to adjust the pH value to 6-7, standing, and adding ammonia water or a NaOH solution to adjust the pH value to 8-9 to obtain the immunogen.

Further, in step S2, the first immunization is performed by mixing and emulsifying the immunogen and the complete freund adjuvant, the multiple booster immunization is performed by mixing and emulsifying the immunogen and the incomplete freund adjuvant, and the last immunization is performed by injecting the immunogen into the abdominal cavity after diluting the immunogen with normal saline.

Specifically, the first immunization dose is 100 mug/mouse; the multiple booster dose is 50 mug/mouse; the interval between the first immunization and the second boosting immunization is one month, and the interval between the multiple boosting immunizations is 21 days; the interval between the sprint immunization and the last booster immunization is 18-21 days.

In the step S3, the mouse spleen cells and mouse myeloma cells selected in the step S2 are fused by a polyethylene glycol (PEG 4000) method, the fusion is carried out by a selective medium (HAT medium), positive cell holes are detected by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), the inhibition effect of the positive cell holes is further determined by the ic-ELISA, the positive cell holes with the best inhibition effect are subcloned for three times by a limiting dilution method, and finally, the digitoxin monoclonal antibody hybridoma cell strain is obtained by screening.

The third aspect of the invention provides a digitoxin monoclonal antibody, which is secreted and generated by the digitoxin monoclonal antibody hybridoma cell strain FJN with the preservation number of CGMCC No. 22325.

The fourth aspect of the invention provides the application of the digoxigenin monoclonal antibody in digoxigenin detection.

In particular, the method can be used for monitoring the digitoxin concentration in clinical blood samples.

The invention also provides a kit for detecting digoxigenin, which comprises the digoxigenin monoclonal antibody; and a kit for detecting digoxigenin, comprising the digoxigenin monoclonal antibody.

Compared with the prior art, the technical scheme of the invention has the following advantages: the invention provides digitalis venomThe monoclonal antibody secreted by the glycoside monoclonal antibody hybridoma cell strain has better specificity and detection sensitivity (IC) on digitoxin₅₀A value of 0.51ng/mL), provides an immunological method for detecting the digoxigenin content in a blood sample clinically. The digoxigenin monoclonal antibody hybridoma cell strain and the monoclonal antibody secreted by the same can be prepared into a kit for detecting digoxigenin, and have practical application values.

Biological material preservation: a digitalis glycosides monoclonal antibody hybridoma cell strain FJN is deposited in China general microbiological culture Collection center (CGMCC), the microbial research institute of China academy of sciences, No. 3, West Lu 1, North Cheng, Xilu, the south China Committee, Beijing, and the monoclonal antibody cell strain is classified and named as a monoclonal cell strain, the preservation date is 2021 year, 05 month and 13 days, and the preservation number is CGMCC No. 22325.

Drawings

FIG. 1 is a standard curve of digoxigenin monoclonal antibody inhibition against digoxigenin.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

The solutions in the following examples were prepared as follows:

carbonate Buffer (CBS): weighing Na₂CO₃1.59 g，NaHCO₃2.93 g, respectively dissolving in a small amount of double distilled water, mixing, adding the double distilled water to about 800mL, uniformly mixing, adjusting the pH value to 9.6, adding the double distilled water to a constant volume of 1000mL, and storing at 4 ℃ for later use.

Phosphate Buffered Saline (PBS): 8.0g NaCl, 0.2g KCl, 0.2g KH₂PO₄，2.9g Na₂HPO₄·12H₂Dissolving O in 800mL of pure water, adjusting the pH value to 7.2-7.4 by using NaOH or HCl, and metering the volume to 1000 mL;

wash solution (PBST): adding 0.5mL of Tween-20 into 1000mL of PBS solution with the concentration of 0.01 mol/LpH7.4;

PBST: PBS containing 0.05% Tween-20;

antibody dilution: wash buffer containing 0.1% gelatin;

TMB color development liquid: solution A: na (Na)₂HPO₄.12H₂18.43g of O, 9.33g of citric acid and pure water to reach the constant volume of 1000 mL; and B, liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. Mixing the liquid B according to the volume ratio of 1:5 to obtain the TMB color developing solution, and mixing the liquid B at the present time.

EXAMPLE 1 preparation of hybridoma cell line FJN

(1) Preparation of the immunogen digoxigenin-BSA:

21.4mg digitoxin was weighed out, dissolved in 1mL dimethyl sulfoxide (DMSO), and 0.1M sodium periodate (NaIO) was added dropwise₄) Stirring the solution for 1mL at room temperature for 1 h; adding 60 μ L of 1M ethylene glycol, and stirring for 5 min; the above mixed reaction solution was added dropwise to a 28mg/mL Bovine Serum Albumin (BSA) solution, and passed through 5% potassium carbonate (K)₂CO₃) Adjusting the pH value to 9, and continuously stirring for 1h until the pH value is stable; weighing 15mg of sodium borohydride (NaBH)₄) Dissolving in water, and adding into the reaction solution to react for 16 h; adding 1M formic acid, adjusting the pH value to 6.5, and standing for 1h at room temperature; adding ammonia water (NH)₄OH), adjusting the pH value to 8.5; dialyzing the reaction solution for 24 hours; after dialysis, 0.1M hydrochloric acid (HCl) was used to adjust pH to 5, and the precipitate was left to stand at room temperature for 1 hour and at 4 ℃ for 3 hours; centrifuging at 12000rpm for 8min, precipitating with sodium bicarbonate (NaHCO)₃) Dissolving, and separating complete antigen and unconjugated small molecule hapten by dialysis to obtain immunogen digoxigenin-BSA.

(2) Preparation of coated protopanoxadiol digoxigenin-OVA:

21.4mg digitoxin was weighed out, dissolved in 1mL dimethyl sulfoxide (DMSO), and 0.1M sodium periodate (NaIO) was added dropwise₄) Stirring the solution for 1mL at room temperature for 1 h; adding 60 μ L of 1M ethylene glycol, and stirring for 5 min; the above mixed reaction solution was added dropwise to a 28mg/mL egg albumin (OVA) solution, and passed through 5% potassium carbonate (K)₂CO₃) Adjusting the pH value to 9, and continuously stirring for 1h until the pH value is stable; weighing 15mg of sodium borohydride (NaBH)₄) Dissolving in water, and adding into the reaction solution to react for 16 h; adding 1M formic acid, adjusting the pH value to 6.5, and standing for 1h at room temperature; adding ammonia water (NH)₄OH), adjusting the pH value to 8.5; dialyzing the reaction solution for 24 hours; after dialysisAdjusting pH to 5 with 0.1M hydrochloric acid (HCl), standing the precipitate at room temperature for 1h, and standing at 4 ℃ for 3 h; centrifuging at 12000rpm for 8min, precipitating with sodium bicarbonate (NaHCO)₃) Dissolving, and separating complete antigen and unconjugated small molecule hapten by dialysis to obtain immunogen digoxigenin-OVA.

(3) Animal immunization: healthy 6-8 week old BALB/c mice were selected for immunization. The complete antigen of digoxigenin (immunogen digoxigenin-BSA) is mixed and emulsified with equal volume of Freund's adjuvant, and then BALB/c mice are immunized by subcutaneous injection into the back. Complete Freund's adjuvant is used for the first immunization, and incomplete Freund's adjuvant is used for multiple times of boosting immunization. One month is separated between the first immunization and the second boosting immunization, 21 days are separated between multiple boosting immunizations, the booster immunization is carried out by intraperitoneal injection (without adjuvant) after the immunogen is diluted by physiological saline for the last time, and 18-21 days are separated between the booster immunization for the last time. The serum titer and inhibition of the mice were determined by ic-ELISA, and mice with high titer and good inhibition were selected.

(4) Cell fusion: after three days of the spurting immunization, cell fusion is carried out according to a conventional PEG (polyethylene glycol, molecular weight is 4000) method, and the specific steps are as follows:

a. taking eyeballs and blood, immediately putting the mice into 75% alcohol for disinfection after the mice are killed by a cervical vertebra dislocation method, soaking for about 5min, taking out the spleen of the mice by aseptic operation, properly grinding the spleen by using a rubber head of an injector, passing through a 200-mesh cell screen to obtain a spleen cell suspension, collecting, centrifuging (1200rpm, 8min), washing the spleen cells for three times by using an RPMI-1640 culture medium, diluting the spleen cells to a certain volume after the last centrifugation, and counting for later use;

b. collecting SP2/0 cells: 7-10 days before fusion, SP2/0 tumor cells were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) at 5% CO₂An incubator. Before fusion, SP2/0 tumor cell number is required to reach 1-4 x 10⁷Ensuring that SP2/0 tumor cells are in logarithmic growth phase before fusion. During fusion, tumor cells are collected and suspended in RPMI-1640 basic culture solution for cell counting;

c. the fusion process is 7 min. 1min, dripping 1mL of PEG into the cells from slow to fast; standing for 2 min. 3min andat 4min, 1mL of RPMI-1640 culture medium is added dropwise within 1 min; dropping 2mL of RPMI-1640 culture medium within 1min at 5min and 6 min; at 7min, 1mL of RPMI-1640 medium was added dropwise every 10 s. Then, the mixture is incubated at 37 ℃ for 5min. Centrifuging (800rpm, 8min), discarding supernatant, lightly tapping cells, resuspending in RPMI-1640 medium containing 20% fetal bovine serum and 2% 50 XHAT, adding to 96-well cell plate at 200. mu.L/well, placing at 37 ℃ and 5% CO₂Culturing in an incubator.

(5) Cell screening and cell strain establishment: on day 3 of cell fusion, the fused cells were subjected to RPMI-1640 screening medium half-replacement, on day 5, to total-replacement with RPMI-1640 medium containing 20% fetal bovine serum and 1% 100 XHT, and on day 7, cell supernatants were collected for screening. The screening is divided into two steps: firstly, screening out positive cell holes by using ic-ELISA, and secondly, selecting digitoxin as a standard substance and measuring the inhibition effect of the positive cells by using ic-ELISA. Selecting cell wells with good inhibition to digoxigenin standard, subcloning by limiting dilution method, and detecting by the same method. This was repeated three times to obtain cell line FJN.

(6) Preparation and identification of monoclonal antibody: taking BALB/c mice 8-10 weeks old, and injecting 1mL of sterile paraffin oil into the abdominal cavity of each mouse; 7 days later, each mouse was injected intraperitoneally with 1X 10⁶Hybridoma cells, ascites fluid was collected from the seventh day, and the ascites fluid was purified by the octanoic acid-ammonium sulfate method. Under the condition of partial acid, the caprylic acid can precipitate other hybrid proteins except IgG immunoglobulin in the ascites, then the centrifugation is carried out, and the precipitate is discarded; then, the IgG type monoclonal antibody was precipitated with an ammonium sulfate solution of the same saturation, centrifuged, the supernatant was discarded, dissolved in a 0.01M PBS solution (pH 7.4), dialyzed and desalted to finally obtain a purified monoclonal antibody, which was stored at-20 ℃.

Example 2 IC of Didigoxin monoclonal antibody₅₀Measurement of (2)

(1) Coating: the coated digoxigenin-OVA was diluted in 0.05M carbonate buffer pH 9.6 at 1. mu.g/mL in duplicate, 100. mu.L/well, and reacted at 37 ℃ for 2 h.

(2) Washing: the plate solution was decanted and washed 3 times for 3min each with washing solution.

(3) And (3) sealing: after patting to dryness, 200. mu.L/well blocking solution was added and reacted at 37 ℃ for 2 hours. And drying after washing for later use.

(4) Sample adding: diluting antiserum (which is obtained by diluting the antiserum by a corresponding multiple with an antibody diluent after tail-cutting blood collection of a mouse) from 1:1000 by multiple, adding the diluted antiserum into coated holes of each dilution, reacting at the temperature of 100 mu L/hole for 30 min; after washing sufficiently, HRP-goat anti-mouse IgG diluted at a ratio of 1:3000 was added thereto at 100. mu.L/well, and the reaction was carried out at 37 ℃ for 30 min.

(5) Color development: the ELISA plate was removed, washed thoroughly, and 100. mu.L of TMB developing solution was added to each well, and the reaction was carried out at 37 ℃ in the dark for 15min.

(6) Termination and measurement: the reaction was stopped by adding 50. mu.L of a stop solution to each well, and the OD (450nm) of each well was measured by a microplate reader.

Determination of IC of the monoclonal antibody digoxigenin by IC-ELISA₅₀Is 0.51ng/mL, which shows that the reagent has good sensitivity to digoxigenin and can be used for immunoassay detection of digoxigenin.

The cross-reaction of digitoxin monoclonal antibody to digitoxin, digoxin, acetyldigitoxin and ouabain was determined separately and the results are shown in table 1:

TABLE 1 specificity of monoclonal antibody 4B12

Note: cross-reactivity (CR) IC of digoxigenin₅₀Structural analogue IC₅₀*100％。

The result shows that the digitoxin monoclonal antibody has extremely low cross reaction rate to digoxin, acetyl digitoxin and ouabain, so that the antibody prepared by the invention has better specificity to digitoxin.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims

1. A digitalis glycosides monoclonal antibody hybridoma cell strain FJN is preserved in China general microbiological culture Collection center (CGMCC), China academy of sciences, China institute of microbiology, No. 3, West Lu 1 institute of North Chen West Lu, south China Committee, Beijing, with a preservation date of 2021 year, 05 month and 13 days, and the preservation number is CGMCC No. 22325.

2. A method for producing the digoxigenin monoclonal antibody hybridoma cell line FJN according to claim 1, comprising the steps of,

s1: coupling digitoxin with carrier protein to obtain immunogen;

3. The method according to claim 2, wherein the carrier protein is bovine serum albumin or keyhole limpet hemocyanin.

4. The production method according to claim 2 or 3, wherein the step S1 is specifically performed as follows,

SS 3: and adding sodium borohydride, and reacting to obtain the immunogen.

5. The process according to claim 4, wherein in 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.

6. The method of claim 2, wherein in step S2, the first immunization is performed by mixing and emulsifying the immunogen with complete freund 'S adjuvant, the multiple booster immunizations are performed by mixing and emulsifying the immunogen with incomplete freund' S adjuvant, and the last immunization is performed by performing the stab immunization by intraperitoneal injection after diluting the immunogen with physiological saline.

7. The digitoxin monoclonal antibody is characterized by being secreted and produced by a digitoxin monoclonal antibody hybridoma cell strain FJN with the preservation number of CGMCC No. 22325.

8. Use of the digoxigenin monoclonal antibody of claim 7 in digoxigenin detection.

9. A composition for detecting digoxigenin, comprising the digoxigenin monoclonal antibody of claim 7.

10. A kit for detecting digoxigenin, comprising the digoxigenin monoclonal antibody of claim 7.