Hybridoma cell strain secreting ceftazidime monoclonal antibody and application thereof

Abstract

The invention relates to a hybridoma cell strain secreting a ceftazidime monoclonal antibody and application of the hybridoma cell strain, and belongs to the technical field of immunodetection. A hybridoma cell strain capable of accurately detecting low-concentration ceftazidime is separated, specifically, a monoclonal antibody secreted by the hybridoma cell strain has good sensitivity and specificity to the ceftazidime, the IC50 value of the monoclonal antibody to the ceftazidime is 0.40 ng / mL, the IC50 value of the monoclonal antibody to the ceftazidime is 0.40 ng / mL, and the IC50 value of the monoclonal antibody to a structural analogue of the ceftazidime is 0.40 ng / mL. According to the present invention, ceftazidime with low concentration can be accurately detected, such as cefazolin, cefradine, cefalexin, cefquinoline and cefalotin, no cross reaction exists, and the specificity is good, such that the low-concentration ceftazidime can be accurately detected.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunoassay, in particular to a hybridoma cell strain secreting ceftazidime monoclonal antibody and application thereof. BACKGROUND

[0002] Ceftazidime is the English common name of the third generation of cephalosporin antibiotics, which is famous for its strong activity against pseudomonas aeruginosa. At the same time, it has excellent effect on most gram-negative bacteria (G-), but not as good as the first and second generation of cephalosporins on gram-positive bacteria (G+). It is clinically used to treat severe infections caused by sensitive bacteria, especially hospital-acquired infections. It is often used for infections in patients with low immune function (such as after chemotherapy, after organ transplantation). Key indications include septicemia, meningitis, complex urinary tract infection, lower respiratory tract infection (such as pneumonia), skin and soft tissue infection, intra-abdominal infection, etc., especially when the pathogen is suspected or confirmed to be pseudomonas aeruginosa. The mechanism of action of ceftazidime is the same as that of other β-lactam antibiotics, mainly acting on the cell wall of bacteria. As a clinical drug, microbiological detection, therapeutic drug monitoring and safety monitoring are necessary means to ensure drug safety. Therefore, it is necessary to accurately quantify the ceftazidime in biological samples.

[0003] At present, the detection method of ceftazidime mainly uses instrument detection, and the commonly used methods are gas chromatography, liquid chromatography and gas chromatography-mass spectrometry. Although these chromatography-based methods have high sensitivity and specificity, there are some disadvantages, such as the need for thorough sample purification, high solvent consumption, expensive equipment and skilled technicians. Therefore, a rapid and simple method for detecting ceftazidime residues is needed.

[0004] Enzyme-linked immunosorbent assay (ELISA) is a highly efficient, sensitive and rapid detection method. The sample pretreatment is simple, the purification steps are few, the analysis capacity is large, the detection cost is low, and the operation is simple, which is suitable for on-site rapid detection of a large number of samples, and has been widely used in drug residue analysis. The premise of using enzyme-linked immunosorbent assay to detect ceftazidime is to obtain a monoclonal antibody with high specificity and high sensitivity to ceftazidime, and there is no hybridoma cell strain that can secrete ceftazidime monoclonal antibody at present. SUMMARY

[0005] Therefore, the technical problem to be solved by the present application is to overcome the problem that there is no hybridoma cell strain that can secrete ceftazidime monoclonal antibody in the prior art.

[0006] To solve the above technical problems, the application provides a hybridoma cell strain secreting ceftazidime monoclonal antibody and application thereof. 50 The hybridoma cell strain of the application secretes the monoclonal antibody with good sensitivity and specificity to ceftazidime, and the IC

[0007] The first object of the application is to provide a hybridoma cell strain, which is preserved in the China General Microbiological Culture Collection Center on April 17, 2025, and classified and named as a monoclonal cell strain, and the preservation address is No. 3, Beichen West Road, Beijing City, and the preservation number is CGMCC No. 46515.

[0008] Further, the hybridoma cell strain is obtained by immunizing animals with a complete antigen prepared from a hapten, wherein the structure of the hapten is shown as formula I.

[0009]

[0010] Further, the complete antigen is obtained by coupling the hapten with a carrier protein.

[0011] Further, the carrier protein includes keyhole limpet hemocyanin.

[0012] The second object of the application is to provide application of the hybridoma cell strain in detecting ceftazidime.

[0013] The third object of the application is to provide a monoclonal antibody secreted by the hybridoma cell strain.

[0014] The fourth object of the application is to provide application of the monoclonal antibody in detecting ceftazidime.

[0015] The fifth object of the application is to provide a detection product of ceftazidime, which includes the monoclonal antibody.

[0016] Further, the detection product further includes a coating agent.

[0017] Further, the coating agent is prepared by coupling a hapten with a carrier protein, and the carrier protein includes chicken egg white protein.

[0018] The application has the following beneficial effects:

[0019] The application discloses a hybridoma cell strain secreting ceftazidime monoclonal antibody and an application thereof. 50 The IC50 value of the monoclonal antibody secreted by the hybridoma cell strain of the application to ceftazidime is 0.40 ng / mL, and the monoclonal antibody of the application has no cross reaction to structural analogues of ceftazidime, such as ceftizoxime, ceftalozide, cephalexin, cefquinome and cephalothin, has good specificity, and therefore can accurately detect low-concentration ceftazidime.

[0020] Biological material preservation

[0021] The hybridoma cell strain CAZ is preserved in the China General Microbiological Culture Collection Center on April 17, 2025, is classified and named as a monoclonal cell strain, has a preservation number of CGMCC No. 46515, and has a preservation address of No. 3, Beichen West Road, Beijing City, Chaoyang District. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to make the content of the application more easily understood, the application is further described in detail below according to specific embodiments of the application and in combination with the drawings, in which

[0023] Figure 1 The figure is a standard curve of the ceftazidime monoclonal antibody of the application to ceftazidime. DETAILED DESCRIPTION

[0024] The application is further described below in combination with the drawings and specific embodiments, so that those skilled in the art can better understand the application and implement the application, but the embodiments are not used as a limitation to the application.

[0025] The culture medium involved in the following embodiments is as follows.

[0026] 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, magnesium sulfate anhydrous 48.84, sodium phosphate monobasic anhydrous 676.13, potassium chloride 400, sodium chloride 6000, glucose 2000, reduced glutathione 1, phenol red 5, L-glutamine 300, biotin 0.2, calcium D-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 0.005, sodium bicarbonate 2000.

[0027] The reagents involved in the following examples are as follows:

[0028] Carbonate buffer solution (CBS): weigh Na2CO3 1.59 g and NaHCO3 2.93 g, respectively, dissolve in a small amount of double distilled water, mix, add double distilled water to about 800 mL, mix well, adjust the pH value to 9.6, add double distilled water to 1000 mL, store at 4°C for standby use.

[0029] Phosphate buffer solution (PBS): 8.00 g NaCl, 0.2 g KCl, 0.2 g KH2PO4, 2.9 g Na2HPO4·12H2O, dissolved in 800 mL of pure water, adjusted to pH 7.2-7.4 with NaOH or HCl, and diluted to 1000 mL;

[0030] PBST: PBS containing 0.05% Tween 20;

[0031] Antibody diluent: PBS containing 0.1% gelatin;

[0032] TMB developing solution: A solution: Na2HPO4·12H2O 18.43 g, citric acid 9.33 g, pure water to 1000 mL; B solution: 60 mg TMB dissolved in 100 mL ethylene glycol. A and B solutions are mixed at a ratio of 5:1 to obtain TMB developing solution, which is mixed before use.

[0033] The detection method involved in the following examples is as follows:

[0034] Ceftazidime inhibition rate detection method: The most appropriate antigen and antibody concentrations in ic-ELISA were selected by checkerboard test. The antigen was diluted to 0.01, 0.03, 0.1 and 0.3 μg / mL with carbonate buffer solution (CBS), and the antibody was diluted to 0.03, 0.1, 0.3 and 1 μg / mL with antibody diluent. After selecting the optimal working point, the ceftazidime standard was diluted to 8 concentrations (0, 0.02, 0.05, 0.1, 0.2, 0.5, 1 and 2 ng / mL), and the ic-ELISA operation steps were followed. Finally, the OriginPro 8.5 was used for plotting (the results are shown in Figure 1 50 .

[0035] Example 1: Synthesis of ceftazidime complete antigen

[0036] Since ceftazidime itself contains a functional group of amino, it has immunogenicity and can stimulate the immune response of mice to produce antibodies, so it is only necessary to directly couple the amino group with the protein of the carrier.

[0037] Weigh 14.8 mg of ceftazidime into a small amount of DMSO, add 81 μL of 1M HCL, and ice water bath for 30 min. Then add 9.32 μL of 30% NaNO2, continue to stir for 30 min for activation. Take 6 mg of keyhole limpet hemocyanin (KLH), add to 3 mL of 0.01M carbonate buffer solution (CBS), and dissolve thoroughly. Slowly add the activated solution to the diluted KLH solution, stir overnight at room temperature. Then dialyze with 0.01M PBS solution to remove unreacted small molecules, obtain relatively pure complete antigen, and identify by ultraviolet absorption scanning method.

[0038] Example 2: Synthesis of ceftazidime coating antigen

[0039] Weigh 6.56 mg of ceftazidime into a small amount of DMSO, add 36 μL of 1M HCL, and ice water bath for 30 min. Then add 4.14 μL of 30% NaNO2, continue to stir for 30 min for activation. Dissolve 6 mg of chicken ovalbumin (OVA) in carbonate buffer solution (CBS); slowly add the activated solution to the protein diluent, stir overnight at room temperature. Then dialyze the reaction solution with 0.01M PBS solution to remove unreacted small molecules, obtain the coating antigen.

[0040] Example 3: Preparation of hybridoma cell strain secreting ceftazidime monoclonal antibody

[0041] 1. Obtaining of animal immunization

[0042] ​The cefotetan complete antigen is mixed with an equal amount of Freund's adjuvant and emulsified, and then the BALB / c mice are immunized by subcutaneous injection at multiple points on the back of the neck (except for the booster immunization); the first immunization is performed using complete Freund's adjuvant at a dose of 100 μg per mouse; the multiple booster immunizations are performed using incomplete Freund's adjuvant at a dose of 50 μg per mouse; the booster immunization is performed using physiological saline without adjuvant at a dose of 25 μg per mouse; the interval between the first immunization and the second booster immunization is one month, the interval between the multiple booster immunizations is 21 days, and the interval between the booster immunization and the last booster immunization is 18-21 days; the immune effect of the mice is observed by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to detect the titer and inhibition of the serum of the mice;

[0043] 2. Cell fusion

[0044] Three days after the booster immunization, the cell fusion is performed according to the conventional PEG (polyethylene glycol, molecular weight 4000) method, and the specific steps are as follows:

[0045] a. Tail bleeding, after the mice are sacrificed by cervical dislocation, they are immediately sterilized in 75% alcohol for about 5 min, the spleen of the mouse is removed by sterile operation, and the spleen cell suspension is obtained by moderately grinding the spleen with a syringe rubber head and passing through a 200-mesh cell sieve, collected, centrifuged (1200 rpm, 8 min), and the spleen cells are washed three times with RPMI-1640 medium, and finally diluted to a certain volume, counted, and reserved;

[0046] b. Collection of SP2 / 0 cells: 7-10 days before fusion, the SP2 / 0 tumor cells are expanded in an RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator, and the number of SP2 / 0 tumor cells is required to reach (1-4) x 10 7 before fusion to ensure that the SP2 / 0 tumor cells are in the logarithmic growth phase before fusion, and the tumor cells are collected and suspended in RPMI-1640 basic culture medium for cell counting;

[0047] c. Fusion process 7 min: 1 mL of PEG 4000 was added slowly to fast into the cells in the first minute; stand for 2 minutes; 1 mL of RPMI-1640 medium was added in 1 minute in the third and fourth minutes; 2 mL of RPMI-1640 medium was added in 1 minute in the fifth and sixth minutes; 1 mL of RPMI-1640 medium was added every 10 seconds in the seventh minute. The solution was constantly shaken except for the second minute. Then it was incubated at 37°C for 5 minutes; centrifuged (800 rpm, 8 minutes), the supernatant was discarded, and it was resuspended in RPMI-1640 screening medium containing 20% fetal bovine serum and 2% 50x HAT, added to a 96-well cell plate at 200 μL / well, and incubated at 37°C in a 5% CO2 incubator.

[0048] 3. Cell screening and cell strain establishment

[0049] The fusion cells were semi-replaced with RPMI-1640 screening medium on the third day after cell fusion, fully replaced with RPMI-1640 transition medium containing 20% fetal bovine serum and 1% 100x HT on the fifth day, and the cell supernatant was taken for screening on the seventh day.

[0050] The screening was divided into two steps: first, positive cell wells were screened by ic-ELISA; second, the positive cells were subjected to inhibition effect determination by ic-ELISA using ceftazidime as a standard.

[0051] The cell wells with good inhibition of the ceftazidime standard were subjected to subcloning by limited dilution, and the same method was used for detection after seven days.

[0052] The subcloning was performed at least three times according to the above method, and the ceftazidime monoclonal antibody cell strain was finally obtained.

[0053] Example 4: Preparation and identification of ceftazidime monoclonal antibody

[0054] 8-10-week-old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil; 7 days later, each mouse was injected intraperitoneally with 1 x 10 6 Ceftazidime hybridoma cells were collected from the ascites starting from the seventh day, and the ascites was subjected to antibody purification by the caprylic acid-saturated ammonium sulfate method.

[0055] In an acidic condition, n-octanoic acid can precipitate other impurities in the ascites except IgG immunoglobulin, and then centrifuged and discarded the precipitate; then the IgG type monoclonal antibody was precipitated by an equal amount of saturated ammonium sulfate solution, centrifuged, and discarded the supernatant; after being dissolved in 0.01 M PBS solution (pH 7.4), it was dialyzed and desalted, and finally the purified monoclonal antibody was stored at -20°C.

[0056] The IC50 of the ceftazidime monoclonal antibody was measured using indirect competitive ELISA, and the result was 0.40 ng / mL. 50 The result showed that the antibody had good sensitivity to ceftazidime and could be used for the immunoassay of ceftazidime.

[0057] The IC50 of the ceftazidime monoclonal antibody to analogues (ceftizoxime, ceftobid, cefalexin, cefquinome, and cephalothin) was verified. 50 The cross-reactivity of the antibody to the analogues was less than 1%, and the cross-reactivity was calculated as follows: cross-reactivity = (IC50 of ceftazidime / IC50 of the analogue) x 100%. According to the cross-reactivity, it can be seen that the antibody had high sensitivity and specificity to ceftazidime, and the details are shown in Table 1. 50 50

[0058] Table 1. IC50 and cross-reactivity of the monoclonal antibody to ceftazidime and analogues 50

[0059] analyte IC 50 (ng / mL) cross-reactivity (%) ceftazidime 0.40 100 cefazolin ＞100 ＜1 cefradine ＞100 ＜1 cephalexin ＞100 ＜1 cefoxitin ＞100 ＜1 cephalothin ＞100 ＜1

[0060] Example 5: Application of the ceftazidime monoclonal antibody

[0061] The monoclonal antibody prepared by the ascites of the hybridoma cell line was applied to the ELISA recovery test of ceftazidime, and the specific steps were as follows:

[0062] (1) The coating antigen diluted with carbonate buffer (CBS) at a concentration of 0.1 μg / mL was coated on a 96-well enzyme-labeled plate at 100 μL per well, and then the plate was baked at 37°C for 2 h. Then, the plate was washed three times with PBST at 200 μL per well, and each washing lasted for 3 min. After washing, the plate was dried.

[0063] (2) The plate was blocked with 0.2% gelatin-containing CBS at 200 μL per well, and then the plate was baked at 37°C for 2 h. Then, the plate was washed three times with PBST at 200 μL per well, and each washing lasted for 3 min. After washing, the plate was dried.

[0064] (3) The phosphate buffer (PBS) was used to prepare 0, 0.02, 0.05, 0.1, 0.2, 0.5, 1, and 2 ng / mL standard solutions of ceftazidime, respectively. The standard solutions and the sample extract to be detected were added to the blocked enzyme-labeled plate at 50 μL per well, and each sample was repeated in three wells. Then, 50 μL of the ceftazidime monoclonal antibody diluted to 0.1 μg / mL was added to each well, and the plate was reacted at 37°C for 30 min. After reaction, the plate was washed and dried.

[0065] (4) 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody diluted to 1:3000 with 0.1% gelatin-containing PBS was added to each well, and the plate was reacted at 37°C for 30 min. After reaction, the plate was washed and dried.​​​

[0066] (5) Add 100 μL TMB color developing solution to each well, color develop at 37°C for 15 min, then add 50 μL 2M H2SO4 stop solution to each well, and measure the absorbance at 450 nm;

[0067] (6) Recovery:

[0068] Urine was selected as the detection sample.

[0069] Cefotetan standard was added to the sample at concentrations of 0.02 ppb, 0.2 ppb, and 2 ppb (according to the linear range of the antibody and IC 50 Set the concentration of the added sample, and mix well on an electric shaker. The recovery rate was 91%, 94%, and 92% respectively when the indirect competitive ELISA was used for the recovery test.

[0070] Obviously, the above examples are merely examples for the sake of clarity, and are not limiting of the embodiments. Other different forms of changes or variations can be made by those of ordinary skill in the art based on the above description. It is not necessary or possible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A hybridoma cell line, characterized in that, The hybridoma cell line was named a monoclonal cell line and its accession number was CGMCC No. 46515.

2. The hybridoma cell line according to claim 1, characterized in that, The hybridoma cell line was obtained by immunizing animals with a complete antigen prepared from a hapten, wherein the structural formula of the hapten is shown in Formula I:

3. The hybridoma cell line according to claim 2, characterized in that, The complete antigen is obtained from the hapten coupled with the carrier protein.

4. The hybridoma cell line according to claim 3, characterized in that, The carrier protein includes keyhole hemocyanin.

5. The use of the hybridoma cell line according to any one of claims 1-4 in the detection of ceftazidime.

6. A monoclonal antibody secreted by a hybridoma cell line according to any one of claims 1-4.

7. The use of the monoclonal antibody according to claim 6 in the detection of ceftazidime.

8. A ceftazidime detection product, characterized in that, The detection product includes the monoclonal antibody as described in claim 6.

9. The testing product according to claim 8, characterized in that, The tested products also include coating agents.

10. The testing product according to claim 9, characterized in that, The coating is prepared from a hapten-coupled carrier protein, wherein the carrier protein includes chicken ovalbumin.

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