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Separation method of Streptococcus pneumoniae in complex matrix

A Streptococcus pneumoniae and complex matrix technology, applied in the biological field, can solve the problems of separation failure, high concentration of miscellaneous bacteria, complex matrix properties, etc., and achieve the effect of increasing the chance of contact, improving the separation efficiency, and shortening the separation time

Inactive Publication Date: 2015-08-12
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 matrices; 3) micron magnetic beads have poor monodispersity, and are prone to self-aggregation or precipitation in sputum and other matrix fluids ; 4) Directly coupling antibody molecules to immunomagnetic beads, this process often leads to greatly reduced antibody activity and changes in the spatial direction of antibodies, increasing the steric hindrance effect between antibodies, thereby reducing the antibody's activity Capture 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 Too high 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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  • Separation method of Streptococcus pneumoniae in complex matrix
  • Separation method of Streptococcus pneumoniae in complex matrix

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 3.6 mg dendrimer aminated polyamide-amine dendrimer in 2 mL, 0.02 M, pH 6.5 PBS, add 0.6 mg NHSS, 0.4 mg EDC, place on a mixer at room temperature Stir and activate for 15 min;

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

[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 15 mg long-chain biotin, 3.6 mg NHSS, and 2.4 mg EDC in 2 mL 0.02 M pH 6.5 PBS buffer;

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

Embodiment 2

[0040] Example 2 Enrichment effect experiment

[0041] (1) Take 1 mL of concentration as 10 4 cfu / mL Streptococcus pneumoniae in a 1.5 mL sterile centrifuge tube, centrifuge at 12,000 rpm for 5 min, 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 with 1 mL of sterile PBS solution Immunomagnetic bead complexes.

[0044] (4) Capture rate calculation: After grad...

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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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 pneumoniae , 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 infection...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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