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Method for separating streptococcus pneumoniae in complicated substrate

A Streptococcus pneumoniae and complex matrix technology, applied in the biological field, can solve the problems of separation failure, increase in antibody spatial direction, cell extrusion, etc., to increase the chance of contact, realize cascade amplification, and stabilize the reaction solution.

Inactive Publication Date: 2013-09-04
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this separation technology has many limitations: 1) The specific surface area of ​​micron magnetic beads is relatively small, which reduces the capture efficiency of magnetic beads; (Multiphase reaction) combination, it usually takes longer to specifically capture bacterial cells in sputum and other substrates; 3) Micron magnetic beads have poor monodispersity, and are prone to self-aggregation or precipitation in sputum and other substrate fluids; 4) The antibody molecules are directly coupled to the immunomagnetic beads. This process often leads to a great reduction in the activity of the antibody and a change in the spatial direction of the antibody, which increases the steric hindrance effect between the antibodies, thereby reducing the capture of the antibody. Efficiency 5) Due to the complex nature of the matrix and the high concentration of non-target pathogenic bacteria, micron magnetic beads are prone to non-specific adsorption, and it is difficult to achieve specific separation of target bacteria in the sample liquid; 6) The concentration of micron magnetic beads is too high High levels will cause damage to bacterial cells (the magnetic field causes the magnetic beads on the cell surface to attract each other, causing the cells to be squeezed or even ruptured), resulting in failure of separation
The distance between the antibody and the surface of the magnetic bead is too close, the nature of the magnetic bead itself and the residual hydrophobic or strong hydrophilic groups on the surface are likely to cause changes in the spatial conformation of the antibody, resulting in a decrease in the biological activity of the antibody

Method used

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  • Method for separating streptococcus pneumoniae in complicated substrate
  • Method for separating streptococcus pneumoniae in complicated substrate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] experiment one:

[0030]1. The dendritic polymer-antibody complex is prepared according to the following steps:

[0031] (1) Dissolve 3.6mg of dendrimer aminated polyamide-amine dendrimer in 2mL, 0.02M, pH6.5PBS, add 0.6mgNHSS, 0.4mgEDC, stir on a mixer at room temperature, activate 15min;

[0032] (2) Take 10.5mg SP The specific antibody was added to the above reaction solution, placed on a mixer at room temperature and stirred for 30 minutes;

[0033] (3) After the above solution was vacuum-dried, dissolved in deionized water, dialyzed in PBS and deionized water for 1 day; after the dialysis, the obtained solution was freeze-dried.

[0034] 2. The long-chain biotin-dendrimer-antibody complex is prepared according to the following steps:

[0035] (1) Dissolve 15mg of long-chain biotin, 3.6mg of NHSS, and 2.4mg of EDC in 2mL of 0.02MpH6.5PBS buffer;

[0036] (2) Add 0.55 mg of the dendrimer-antibody complex to the above solution, place it on a mixer at room temper...

Embodiment 2

[0040] Embodiment 2 enrichment effect experiment

[0041] (1) Take 1mL concentration as 10 4 cfu / mL Streptococcus pneumoniae in a 1.5mL sterile centrifuge tube, centrifuge at 12000rpm for 5min, discard the supernatant, and resuspend with an equal volume of sterile PBS solution.

[0042] (2) Enrichment and capture: Set up the technical solution group of the present invention (dendritic polymer group co-modified by Streptococcus pneumoniae antibody and long-chain biotin), nanomagnetic beads group modified by Streptococcus pneumoniae specific antibody, Streptococcus pneumoniae Specific antibody-modified micron magnetic bead group enriches target bacteria.

[0043] (3) After magnetic separation, pour the supernatant into a sterile centrifuge tube, and wash the isolated immunomagnetic beads with Streptococcus pneumoniae twice with PBST, mix well, and resuspend the immunomagnetic beads with 1 mL of sterile PBS solution. Magnetic bead complex.

[0044] (4) Capture rate calculation...

Embodiment 3

[0057] Example 3 Enrichment capture experiment

[0058] Conventional magnetic stand separation time is 30min, and all the other are with embodiment 2.

[0059] The catch rate of each group is as follows:

[0060] Capture efficiency of micron magnetic bead set modified with specific antibody of Streptococcus pneumoniae Capture efficiency of nano-magnetic bead set modified with specific antibody of Streptococcus pneumoniae Capture efficiency of dendrimers co-modified with Streptococcus pneumoniae antibody and long-chain biotin 46.5% 40.6% 90.9%

[0061] The experimental results show that compared with the separation of 3min in Example 2, when the separation time reaches 30min, the capture efficiency of the three groups has been improved, especially the capture efficiency of the nano-magnetic bead group modified by the specific antibody of Streptococcus pneumoniae is the most obvious. This indicated that the capture efficiency of the magnetic nanobeads gr...

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PUM

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Abstract

The invention discloses a method for enriching and separating streptococcus pneumoniae in order to provide a basis for the follow-up study of target bacteria, relating to the field of biotechnology. The method comprises the steps of: covalent coupling between a dendritic polymer and an antibody, exposed amino of long-chain biotin blocking antibody-dendritic polymer, capturing of target bacteria in sample liquid by the antibody coupled with the dendritic polymer, identification of streptavidin coupled with nano magnetic beads and coupling with the long-chain biotinylated dendritic polymer in the sample liquid, magnetic separation and re-suspension of bacteria and the like. The re-suspension liquid can be directly subjected to follow-up analysis; and compared with the traditional magnetic bacterial separation method, the method disclosed by the invention is more suitable for the magnetic separation of bacteria in a complicated substrate, and improves the separation efficiency of the target bacteria in a sample.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for isolating pathogenic bacteria based on nano magnetic beads. Background technique [0002] Streptococcus pneumoniae ( Streptococcus spneumoniae , SP ), as a clinically important pathogen, is widely distributed in nature, and its carrier rate in healthy people is between 5% and 50%, and it is closely related to colonization and infection. SP In addition to causing sinusitis and otitis media, it is also the number one pathogen that causes serious diseases such as meningitis, lobar pneumonia, and sepsis. In addition, it can cause severe neurotoxicity, such as focal neurological deficits, cognitive impairment, and hearing loss. In recent years, 700,000 to 1,000,000 deaths have been caused by Streptococcus pneumoniae worldwide, and its main incidence is concentrated in the elderly and children. In addition, China is one of the countries with the largest number of infectio...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12R1/46
Inventor 许恒毅魏华杨林熊勇华赖卫华徐锋邓省亮
Owner NANCHANG UNIV
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