Preparation method of piperacillin monoclonal antibody and application

A piperacillin monoclonal antibody technology, applied in the biological field, can solve the problems of delayed disease, insufficient guarantee of piperacillin drug efficacy, and influence on treatment effect, etc.

Inactive Publication Date: 2018-08-31
江苏中济万泰生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when patients are treated with piperacillin in the course of clinical treatment, some patients may develop antibodies against this drug
Once a patient develops anti-piperacillin antibodies, the antigen-antibody complex formed by the antibody and piperacillin will neutralize and inhibit the efficacy of piperacillin, so that the efficacy of piperacillin cannot be fully guaranteed
At the same time, the combination of the immune complex formed by the piperacillin drug and its corresponding antibody and human red blood cells will cause red blood cell hemolysis, which will affect the treatment effect in the slightest, delay the disease, and endanger the life safety of the patient in severe cases.

Method used

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  • Preparation method of piperacillin monoclonal antibody and application
  • Preparation method of piperacillin monoclonal antibody and application
  • Preparation method of piperacillin monoclonal antibody and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment l

[0090] Embodiment 1: the preparation of piperacillin monoclonal antibody

[0091] 1.1 Preparation of feeder cells:

[0092] Take 6-10 week-old Balb / c mice, kill them by pulling their necks, soak them in 75% alcohol, and disinfect them for 3-5 minutes; transfer them to a sterile operating table, cut the skin with sterile scissors, and fully expose their peritoneum; Inject 10mL of pre-cooled serum-free 1640 culture solution into a sterile syringe, gently tap the mouse abdominal cavity with the bottom of the tweezers, suck out the culture solution, put it into a 10mL centrifuge tube, and centrifuge at 1200r / min for 10min; discard the supernatant, and use complete culture solution Resuspend the feeder cells, count, and adjust the number of cells to 2×105 / mL, add the feeder cells into a 96-well plate, 100 μL per well, and then put them in 37°C, CO 2 incubator cultivation;

[0093] 1.2 Hybridoma cell fusion and subcloning screening by limiting dilution method

[0094] a. Preparat...

Embodiment 22

[0108] Example 22: Antibody Purification

[0109] 2.1 Pretreatment of the column:

[0110] a. Take out the prepared Protein A / Protein G filler and transfer it into a 50ml centrifuge tube, add an appropriate amount of 20% (volume ratio) ethanol, mix it upside down, mix the filler and transfer it to an empty column;

[0111] b. After filling the empty column, tighten the connector, and manually discharge 20% of the filler from the connector until 1-2ml of 20% ethanol remains at the outlet.

[0112] c. Connect the prepared column to the AKTA system, wait for the system to balance and remove the air.

[0113] 2.2 Preparation of experimental reagents (all reagents should be sterilized with a 0.45 μm filter):

[0114] a.VBS buffer: pH7.2 barbiturate buffer

[0115] Accurately weigh 0.737g of barbiturate, 8.766g of sodium chloride, 0.197g of magnesium sulfate heptahydrate, and 0.033g of anhydrous calcium chloride, dissolve them in 1L of distilled water, adjust the pH to 7.2 with 1...

Embodiment 3

[0135] Example 3: Identification of antibody purity

[0136] 1. Experimental steps

[0137] Take out the sample, add 0.1% proclin-300, filter it with a 0.22 μm filter, and use the BCA method to measure the antibody concentration. The specific operation is as follows:

[0138] a. According to the number of samples, prepare an appropriate amount of BCA working solution with 50 volumes of BCA reagent A plus 1 volume of BCA reagent B (50:1), and mix well. BCA working solution is stable at room temperature for 24 hours.

[0139] b. Completely dissolve the protein standard, take 10 μl and dilute to 100 μl, so that the final concentration is 0.5 mg / ml. What solution is the protein sample in, and what solution should the standard be diluted with. However, for simplicity, the standards can also be diluted with 0.9% NaCl or PBS.

[0140] c. Add 0, 1, 2, 4, 8, 12, 16, and 20 μl of the standard to the standard wells of the 96-well plate, and add the solution used to dilute the standar...

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Abstract

The invention relates to a preparation method of a piperacillin monoclonal antibody and application. A hybridoma cell strain is utilized for preparation. The preparation method comprises the followingsteps: preparing a piperacillin monoclonal antibody, performing antibody purification, performing antibody purity identification and performing antibody titer identification. The prepared piperacillin monoclonal antibody is used for detecting piperacillin drug antibodies. Blood of a detected object serves as a detection sample, if the piperacillin antibody exists in plasma, the antibody is boundto a piperacillin drug antigen on piperacillin treated cells by virtue of anti-IgG+G3d bridging so as to produce agglutination, the antibody cannot pass through a gel space and remains on the upper layer of the gel or is dispersed into the gel under the effect of centrifugal force, and presents a positive reaction; and if any antibody does not exist in the plasma or the cell surface does not contain any drug antigen, agglutination is not produced, and the antibody can pass through the gel space to deposit at the bottom of a micro-column gel hole under the effect of the centrifugal force and presents a negative reaction.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to the preparation of piperacillin monoclonal antibody by hybridoma technology, and the clinical application of the developed monoclonal antibody. Background technique [0002] During clinical treatment, when drugs are used, especially when antibiotics are used, the body may not only develop drug tolerance, but may also produce anti-drug antibodies against the drug. After the body produces anti-drug antibodies, the drugs, anti-drug antibodies and red blood cells will form an immune complex, which will lead to the destruction and removal of red blood cells by the body, causing immune hemolytic anemia, ineffective use of drugs, and endangering the lives of patients. Although the chance of developing anti-drug antibodies is very low, more than 100 drugs have been reported so far to trigger the production of anti-drug antibodies. With the advancement of science and technolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/44C07K1/16G01N33/96G01N33/94
CPCC07K16/44G01N33/9446G01N33/96G01N2430/00
Inventor 赵树铭林裕翔吴明磊
Owner 江苏中济万泰生物医药有限公司
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