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Monoclonal antibody against plectasin and detection kit for plectasin

A detection kit and monoclonal antibody technology, applied in the field of immunology, can solve the problem that the detection method cannot meet the micro-detection of pecomycin samples, and achieve the effects of simple operation, high sensitivity and good repeatability

Active Publication Date: 2018-12-07
GUANGDONG HINAPHARM PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the deficiencies of the above-mentioned prior art, the object of the present invention is to provide a monoclonal antibody and detection kit for plectasin, which aims to solve the problem that the detection method of the prior art cannot meet the requirement of micro-detection of plectasin samples. question

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  • Monoclonal antibody against plectasin and detection kit for plectasin

Examples

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Effect test

Embodiment 1

[0032] Example 1 Preparation of Plectasin Monoclonal Antibody

[0033] 1. Preparation of Plectasin Antigen

[0034] The plectasin antigen is obtained from the engineering bacteria constructed in our laboratory (China Invention Patent Application No. 201710939331.5), and the specific culture method and the fermentation supernatant of the pphaceas antigen can be referred to in this patent; The supernatant was separated by high-performance liquid chromatography, and the purified liquid of plectasin was collected, and dialyzed at low temperature for 24 hours with a 1000Da dialysis bag, during which the dialysate was replaced several times to achieve the purpose of desalination; the liquid in the dialysis bag was collected, Freeze-dry to obtain the pure product of Plectasin (Plectasin antigen); at the same time, collect several main miscellaneous protein liquids respectively, and obtain freeze-dried samples according to the same treatment method, and set aside.

[0035] 2. Animal ...

Embodiment 2

[0067] Example 2 Construction of Plectasin Detection Kit

[0068] An indirect competitive ELISA method was used to establish a detection kit for plectasin. The kit includes: a 96-well plate coated with Plectasin antigen, a monoclonal antibody (primary antibody) to Plectasin, a standard product of Plectasin, and horseradish peroxidase-labeled goat anti-mouse IgG (HRP enzyme-labeled secondary antibody), sample diluent, sample washing solution, TMB chromogenic solution, stop solution.

[0069] 1. Establishment of standard curve

[0070] Antigen coating: Dilute Plectasin to 2 μg / mL with coating solution, add 100 μL / well into a 96-well plate, and place at 4°C overnight (about 12 hours). The next day, the liquid in the wells was discarded, and washed 3 times with PBST, 300 μL / well each time.

[0071] Blocking: add 300 μL / well PBSM, block at 37°C for 2 hours, wash with PBST 3 times, each time 300 μL / well.

[0072] Preparation of standard solution: Dilute plectasin with PBSTM to t...

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Abstract

The invention discloses a monoclonal antibody against plectasin and a detection kit for plectasin. The monoclonal antibody is secreted by an anti-plectasin monoclonal antibody hybridoma 5D11 which ispreserved in China General Microbiological Culture Collection Center on May 30, 2018, with an accession number of CGMCC No. 15790. The kit comprises a 96-well plate coated by a plectasin antigen, theanti-plectasin monoclonal antibody, a plectasin standard substance, horseradish peroxidase-labeled goat anti-mouse IgG, a sample diluent, a sample washing solution, a TMB developing solution and a stopping solution. The anti-plectasin monoclonal antibody secreted by the hybridoma has high specificity and high titer; and the kit can detect trace amounts of plectasin in various samples and has the advantages of simple operation, high sensitivity, good repeatability and the like.

Description

technical field [0001] The invention relates to the technical field of immunology, in particular to a plectasin monoclonal antibody and a detection kit. Background technique [0002] Plectasin (Plectasin) is obtained from the saprophytic ascomycete Pseudomonas nigella ( Pseudoselectania nigrella ), the first fungal defensin isolated from ), has 50%-55% homology with invertebrate defensins, and has no sequence similarity with mammalian α- or β-defensins. Mature plectasin is composed of 40 amino acid defects, consisting of an α-helix and two antiparallel β-sheets, including a stable spatial structure formed by three disulfide bonds. Plectasin has weak antibacterial effect on Gram-negative bacteria, but has obvious killing effect on Gram-positive bacteria, including: Streptococcus, Staphylococcus, Enterococcus, Corynebacterium and Rod. It is particularly noteworthy that plectasin has a very significant bactericidal effect on drug-resistant Staphylococcus aureus (MRSA), and th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/14C12N5/20G01N33/577
CPCC07K16/14G01N33/577
Inventor 梁伟凡周玉岩范惠敏
Owner GUANGDONG HINAPHARM PHARMA CO LTD
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