CN106701686B

CN106701686B - hybridoma cell strain 1A5, secreted anti-sulbactam monoclonal antibody and application

Info

Publication number: CN106701686B
Application number: CN201610700184.1A
Authority: CN
Inventors: 徐丽广; 陈燕妮; 胥传来; 匡华; 刘丽强; 宋珊珊; 吴晓玲; 胡拥明
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2016-08-23
Filing date: 2016-08-23
Publication date: 2020-01-31
Anticipated expiration: 2036-08-23
Also published as: CN106701686A

Abstract

The invention provides hybridoma cell strain 1A5, the secreted anti-sulbactam monoclonal antibody of which has better detection sensitivity and specificity. hybridoma cell strain 1A5, which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 12031.

Description

hybridoma cell strain 1A5, secreted anti-sulbactam monoclonal antibody and application

Technical Field

The invention belongs to the technical field of immunochemistry, and particularly relates to hybridoma cell strains 1A5, a secreted anti-sulbactam monoclonal antibody and application thereof.

Background

Sulbactam (SBT), also called as penicillanic sulfone acid, is an artificially synthesized irreversible competitive β -lactamase inhibitor, has inhibiting effect on β -lactamase produced by gram-positive bacteria and gram-negative bacteria, has slightly weak antibacterial activity, and only has bactericidal effect on gonococcus and acinetobacter when used singly, but can have obvious synergistic effect when used together with penicillin or cephalosporin drugs, so that the antibacterial activity of the sulbactam is greatly improved, and the antibacterial spectrum is also expanded.

At present, the detection method of sulbactam residues in food mainly comprises a spectrophotometry method, a capillary electrophoresis method, a gas-mass combination method, a liquid-mass combination method and a high-efficiency liquid phase method, wherein the detection line of the spectrophotometry method is poor, the range of a capillary electrophoresis detection matrix is limited, in the gas-mass combination method, the sample pretreatment needs derivatization reaction, the operation is complex, the recovery rate is low, the liquid-mass combination method is high in sensitivity and strong in anti-interference capability, but the equipment is expensive, and the pretreatment process is complex.

Disclosure of Invention

Aiming at the problems, the invention provides hybridoma cell strain 1A5, and the secreted anti-sulbactam monoclonal antibody has better detection sensitivity and specificity.

hybridoma cell strain 1A5, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 12031.

The invention also provides an anti-sulbactam monoclonal antibody secreted by the hybridoma cell strain 1A 5.

The invention also provides application of the anti-sulbactam monoclonal antibody secreted by the hybridoma cell strain 1A5 in detection of sulbactam.

And the application of the anti-sulbactam monoclonal antibody secreted by the hybridoma cell strain 1A5 in preparing an immune tool for detecting sulbactam.

The IC50 value of the anti-sulbactam monoclonal antibody secreted by the hybridoma cell strain 1A5 is 6.04 mug/L, and the detection sensitivity is high; has low cross reaction rate with clavulanic acid, tazobactam and ampicillin and good specificity.

Drawings

FIG. 1 is a standard curve for inhibition of monoclonal antibody 1A 5.

FIG. 2 is an electrophoretic image of immunogen and coatingen, lane 1: BSA, lane 2: SBT-OVA, lane 3 SBT-BSA.

Biological preservation Instructions

The hybridoma cell lines used for preservation are classified as follows: monoclonal cell line YH 3; wherein, the hybridoma cell strain 1A5 is named in the laboratory, and the monoclonal cell strain YH3 is named by classification adopted in preservation;

the preservation unit is called as follows: china general microbiological culture Collection center;

the preservation unit is abbreviated as: CGMCC;

the address of the depository: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing;

the preservation date is as follows: 2016, 1 month, 20 days;

the preservation number is: CGMCC No. 12031.

Detailed Description

Example 1

1. Synthesis of immunogens

Weighing 20.88mg (0.089 mmol) of sulbactam technical material, and dissolving in 3mL of anhydrous DMF; adding 33 mu L (0.13 mmol) of tri-n-butylamine, and stirring for ten min under the ice bath condition; adding 17.4 μ L (0.13 mmol) of ethyl butyl chloroformate, and activating under ice bath condition for 40 min; adding the activated liquid into a BSA solution (100 mg BSA dissolved in CBS buffer solution with pH 9.6), and reacting for 4 hours under an ice bath condition; the reaction mixture was dialyzed against 0.01M PBS buffer for three days to remove unreacted small molecules. The synthesis of sulbactam is similar to the synthesis of immunogen, except that the OVA is used instead of the carrier protein BSA. The coupling result of the immunogen and the coating antigen is identified by electrophoresis, and the figure 2 shows that the coupling of the immunogen and the coating antigen is successful.

2. Animal immunization

The method comprises the steps of selecting healthy 6-8 weeks old BALB/c mice for immunization, mixing and emulsifying sulbactam complete antigen and equivalent Freund's adjuvant, injecting immune BALB/c mice subcutaneously through the back, immunizing times with complete Freund's adjuvant with the immune dose of 100 mug/mouse, using incomplete Freund's adjuvant with the immune dose of 50 mug/mouse, separating months between prime and boost, separating 21 days between boost, collecting blood 7 days after triple immunization, measuring the serum titer and inhibition of the mice by using an indirect competitive ELISA method, selecting the mice with high inhibition of the titer, performing impact immunization 18 days after the four immunization, not using the adjuvant, injecting intraperitoneally, and obtaining the immune dose of 20 mug/mouse.

3. Cell fusion

Three days after the impact immunization, cell fusion was performed according to the PEG (polyethylene glycol, molecular weight 1500) method, which specifically comprises the following steps: (1) taking a spleen of a mouse aseptically, grinding the spleen, passing through a 200-mesh cell screen to obtain a spleen cell suspension, and counting cells; (2) collecting SP2/0 cells, suspending in RPMI-1640 basic culture solution, and counting cells; (3) splenocytes and SP2/0 cells were mixed according to 1:10, centrifuging, fusing with 50% PEG for 1 min, adding RPMI-1640 basic culture medium from slow to fast, centrifuging, suspending in RPMI-1640 screening culture medium containing 20% fetal calf serum and 2% 50 XHAT, adding into 96-well cell culture plate, standing at 37 deg.C and 5% CO₂Cultured in an incubator.

4. Cell screening and cell line establishment the fused cells were subjected to half-exchange of RPMI-1640 screening medium on day 3 of cell fusion, to full-exchange with RPMI-1640 medium containing 20% fetal bovine serum and 1% 100 XHT on day 5, and cell supernatants were collected and screened on day 7.

The screening comprises the steps of , screening positive cell holes by using indirect ELISA, selecting sulbactam as a standard substance, measuring the inhibition effect of the positive cells by using indirect competitive ELISA, selecting the cell holes with better inhibition on the sulbactam standard substance, performing subcloning by using a limiting dilution method, detecting by using the same method, repeating for three times, and obtaining the cell strain 1A5 with better inhibition effect, wherein the average result is shown in table 1.

Table 1:

5. preparation and characterization of monoclonal antibodies

Taking BALB/c mice 8-10 weeks old, and injecting 1mL paraffin oil into the abdominal cavity of each mouse; 7 days later, each mouse was injected intraperitoneally with 1X 10⁶And (3) hybridoma cells, collecting ascites from the seventh day, purifying the ascites by an octanoic acid-ammonium sulfate method, and storing the obtained monoclonal antibody at-20 ℃.

A standard curve was generated by indirect competition ELISA and is shown in FIG. 1, IC₅₀Comprises the following steps: 6.04 mu g/L, which shows that the reagent has good sensitivity to sulbactam and can be used for the immunoassay detection of sulbactam.

6. Potency assay for antibodies

Determination of the titer of the monoclonal antibody prepared in step 5:

(1) coating: diluting the coating antigen SBT-OVA by using 0.05M carbonate buffer solution with pH9.6 from 3 mug/mL to 100 uL/hole, and reacting at 37 ℃ for 2 h;

(2) washing: the solution in the plate is poured off, dried, and washed with washing liquid for 3 times, each time for 3 min;

(3) and (3) sealing: after patting dry, 200 uL/well blocking solution was added and the reaction was carried out at 37 ℃ for 2 h. Drying for later use after washing;

(4) sample adding: diluting the antibody from 1:1000 by times, adding the diluted antibody into coated wells of each dilution, reacting at 37 ℃ for 1h at 100 uL/well; after fully washing, adding HRP-goat anti-mouse IgG diluted by 1:3000, 100 uL/hole, and reacting for 1h at 37 ℃;

(5) color development: taking out the ELISA plate, fully washing, adding 100ul of TMB color development solution into each hole, and carrying out a light-shielding reaction at 37 ℃ for 15 min;

(6) termination and measurement: the reaction was stopped by adding 50ul of stop solution to each well, and each was measured by a microplate readerOD of the well₄₅₀A value;

(7) and (4) interpretation of results: by OD₄₅₀The highest dilution factor of the serum corresponding to the value which is more than or equal to 2.1 times of the negative control hole (namely P/N is more than or equal to 2.1) is the ELISA titer of the antibody, and the titer of the antibody is as follows: 8000 at a coating antigen concentration of 3 mg/mL: 1.

7. experiment for recovering antibody

The monoclonal antibody prepared in the step 5 is applied to an ELISA addition recovery test of sulbactam.

The sampled milk is negative through sulbactam detection, 1mL of milk is sucked into a 15mL centrifuge tube, 20ng, 50ng and 100ng of sulbactam are respectively added into the milk, the milk is diluted by 20 times with 0.01M PBS, and an addition recovery test is carried out by adopting indirect ELISA, and the recovery rates are respectively 99.8%, 103.2% and 107.6%.

8. Cross reaction experiment of drug

The monoclonal antibody prepared in step 5 was subjected to a competition experiment with structural analogues of sulbactam, clavulanic acid, tazobactam and ampicillin, and the results are shown in table 2.

Table 2:

the cross rate of the reaction of the monoclonal antibody and other similar drugs is below 10%, and the monoclonal antibody has good specificity, so that the anti-sulbactam monoclonal antibody prepared by the invention can be used for immunodetection of sulbactam.

Solution preparation:

carbonate Buffer (CBS): weighing Na₂CO₃1.59g，NaHCO₃2.93g, 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): 8.00 g NaCl, 0.2 g KCl, 0.2 g KH₂PO₄，2.9 g Na₂HPO₄·12H₂Dissolving O in 800mL of pure water, adjusting the pH value to 7.2-7.4 by NaOH or HCl, and determiningThe volume is 1000 mL;

PBST: PBS containing 0.05% tween 20;

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: 60 mg of TMB was dissolved in 100 mL of ethylene glycol. A. The liquid B is prepared according to the proportion of 1: 5, mixing to obtain the TMB color developing solution which is mixed at present.

The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. That is, all equivalent changes and modifications made according to the content of the claims of the present invention should be within the technical scope of the present invention.

Claims

1, hybridoma cell strain 1A5, which is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 12031.

2. The hybridoma cell line 1A5 of claim 1, secreting an anti-sulbactam monoclonal antibody.

3. The use of the anti-sulbactam monoclonal antibody secreted by the hybridoma cell line 1A5 according to claim 1 in the preparation of an immunological tool for detecting sulbactam.