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Clostridium difficile exotoxin A test kit and monoclonal antibodies therein

A monoclonal antibody, Clostridium difficile technology, applied in the biological field, can solve the problems of reduced curative effect, incompetence, inapplicability of epidemiological investigation and long-term monitoring, and achieve high sensitivity effect

Inactive Publication Date: 2014-07-30
张春华 +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the one hand, Clostridium difficile widely exists in the hospital environment, and as its dormant spores can tolerate a variety of disinfectants commonly used in hospitals, it has become the main pathogen of nosocomial infectious diarrhea after Campylobacter; Azole and vancomycin, as the two main drugs for the treatment of Clostridium difficile infection, their drug resistance is also constantly accumulating and spreading, and the curative effect is decreasing day by day, and for the treatment of relapsed cases, these two drugs are incompetent state
On the other hand, so far, there is no effective and commercialized vaccine available; toxin-based ELISA detection kits are expensive, and it is difficult to perform routine clinical detection of Clostridium difficile, and it is not suitable for large-scale epidemiological investigations and long-term monitoring; therapeutic antibodies as a safe, effective, and inexpensive alternative to antibiotics have a bright future but are still in the exploratory stage

Method used

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  • Clostridium difficile exotoxin A test kit and monoclonal antibodies therein
  • Clostridium difficile exotoxin A test kit and monoclonal antibodies therein
  • Clostridium difficile exotoxin A test kit and monoclonal antibodies therein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Construction of DNA vaccine pJW4303-TcdA-C

[0029] For details, see Examples 1-4 of the Chinese Invention Patent Application Specification with application number 2010101309048.

Embodiment 2293F

[0030] Example 2.293F cell transfection

[0031] 293F cells (purchased from Invitrogen, cat No. R790-07) were prepared with 293F cell culture medium at 37°C, 5% CO 2 Cultivated in a saturated humidity incubator until the logarithmic growth phase,

[0032] The day before transfection, inoculate cells 6~7x10 5 cells / ml, on the day of transfection, take a small amount of cells and count them, the number of cells is about 1x10 6 cells / ml, prepare lipid-DNA complex.

[0033] a. Dilute 30ug pJW4303-TcdA-C plasmid to 1ml with opti-MEM I, and mix gently.

[0034] b. Dilute 60ul 293fectin to 1ml with opti-MEM I, mix gently and incubate at room temperature for 5min.

[0035] c. Add the pJW4303-TcdA-C plasmid dilution prepared in step a to 293fectin and mix gently.

[0036] d. Incubate at room temperature for 20-30mins to obtain DNA / 293fectin.

[0037] Then the DNA / 293fectin was added to the shake flask, transfected for 48-72h, and then the supernatant was collected and stored at -...

Embodiment 3

[0038] Example 3. Establishment of hybridoma cell lines

[0039] Step 1: Animal Immunization

[0040]Seven Balb / c mice were immunized with pJW4303-TcdA-C prepared in Example 1. Immunization by intramuscular injection combined with in vivo gene introduction, ensure that the needle insertion depth is 2 mm, inject the vaccine, observe the injection local uplift, and immediately perform in vivo electrotransfection at the injection site with WJ-2002 live gene introduction instrument (technical parameters: voltage 50V, pulse The number of times is 3 times in front and back, the wave width is 60 ms, and the frequency is 30 Hz), and the electrotransfection is effective when the mouse leg muscles shake. Four times of DNA immunization were carried out at the 0th, 2nd, 4th, and 8th weeks. After the fourth DNA immunization, the crude supernatant of 293F cells transfected with pJW4303-TcdA-C was injected intraperitoneally, and each mouse was injected with 0.2 mg of protein.

[0041] Step...

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Abstract

The invention belongs to the field of biotechnology, and discloses a clostridium difficile exotoxin A test kit and monoclonal antibodies in the clostridium difficile exotoxin A test kit. The kit for testing clostridium difficile exotoxin A comprises two monoclonal antibodies capable of resisting clostridium difficile exotoxin A c-terminus antigens. The two monoclonal antibodies are respectively the monoclonal antibody capable of resisting the clostridium difficile exotoxin A c-terminus antigen and produced by hybridoma with the preservation number of CGMCC No.4979, and the monoclonal antibody labeled by a horse radish peroxidase and produced by hybridoma with the preservation number of CGMCC No. 4980. According to the clostridium difficile exotoxin A test kit provided by the invention, by the two monoclonal antibodies with high affinity, the sensitivity in the detection of clostridium difficile is remarkably improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a Clostridium difficile exotoxin A detection kit and a monoclonal antibody constituting the kit. Background technique [0002] Clostridium difficile is a Gram-positive, spore-forming, obligately anaerobic Clostridium bacterium. Currently, the prevention, diagnosis, and treatment of Clostridium difficile infection are facing many difficulties. On the one hand, Clostridium difficile widely exists in the hospital environment, and as its dormant spores can tolerate a variety of disinfectants commonly used in hospitals, it has become the main pathogen of nosocomial infectious diarrhea after Campylobacter; Zazole and vancomycin, as the two main drugs for the treatment of Clostridium difficile infection, their drug resistance is also constantly accumulating and spreading, and the curative effect is decreasing day by day, and for the treatment of relapsed cases, these two drugs are incompeten...

Claims

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Application Information

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IPC IPC(8): G01N33/577A61K39/40A61P31/04C12N5/20C07K16/12C12R1/91
Inventor 张春华王世霞金柯黄祖瑚卢山
Owner 张春华