Method for enriching and separating helicobacter pylori
A Helicobacter pylori enrichment technology, applied in the biological field, can solve the problems of separation failure, high concentration of miscellaneous bacteria, poor monodispersity of micron magnetic beads, etc., and achieve the effect of increasing contact opportunities, improving separation efficiency, and shortening separation time
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Embodiment 1
[0028] 1. The dendritic hyperbranched polymer-antibody complex is prepared according to the following steps:
[0029] (1) Weigh 4.5 mg of dendritic polyamide-amine aminated by dendritic hyperbranched polymer, dissolve it in 4 mL of phosphate buffer (PBS, 0.01 mol / L, pH 8.0), stir and add 25% ammonium Dialdehyde aqueous solution 545 μL, so that the final concentration of glutaraldehyde is 3%. React at room temperature for 3.5 h at a rotating speed of 150 r / min on a shaker;
[0030] (2) Add Helicobacter pylori dropwise to the above solution HP Specific antibody 12 mg, so that the final concentration reached about 3 mg / mL. React at room temperature for 24 h at the speed of the shaker at 150 r / min;
[0031] (3) The above solution was spin-dried under reduced pressure, dissolved in deionized water, and dialyzed in PBS and deionized water for 1 day; after the dialysis, the obtained solution was freeze-dried.
[0032] 2. The long-chain biotin-dendritic hyperbranched ...
Embodiment 2
[0038] Example 2 Enrichment effect experiment
[0039] (1) Take 1 mL of concentration as 10 4 cfu / mL Helicobacter pylori in a 1.5 mL sterile centrifuge tube, centrifuge at 12000 rpm for 5 min, discard the supernatant, and resuspend with an equal volume of sterile PBS solution.
[0040] (2) Enrichment and capture: Set up the technical scheme group of the present invention (dendritic hyperbranched polymer group co-modified with Helicobacter pylori antibody and long-chain biotin), nano magnetic beads group modified with Helicobacter pylori specific antibody, and Helicobacter pylori Micron magnetic beads modified with bacteria-specific antibodies were used to enrich the target bacteria.
[0041] (3) After magnetic separation, pour the supernatant into a sterile centrifuge tube, and wash the isolated immunomagnetic beads with Helicobacter pylori captured twice with PBST, mix well, and resuspend with 1 mL sterile PBS solution Immunomagnetic bead complexes.
[0042] (4) Capture r...
Embodiment 3
[0055] Example 3 Enrichment capture experiment
[0056] Conventional magnetic stand separation time is 30min, and all the other are with embodiment 2.
[0057] The catch rate of each group is as follows:
[0058] Capture rate of Helicobacter pylori-specific antibody modified micron magnetic bead set Capture efficiency of magnetic nanobeads modified with specific antibodies against Helicobacter pylori Capture efficiency of dendritic hyperbranched polymer groups co-modified with Helicobacter pylori antibody and long-chain biotin 53.1% 34.8% 91.0%
[0059] The experimental results show that compared with the separation of 3 minutes in Example 2, when the separation time reaches 30 minutes, the capture efficiency of the three groups has been improved, especially the capture efficiency of the Helicobacter pylori-specific antibody-modified nano magnetic bead group is the most obvious. This shows that the capture efficiency of the nano-magnetic bead group...
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