Novel method for enriching and separating Candida albicans
An enrichment technology for Candida albicans, applied in the biological field, can solve the problems of separation failure, high concentration of miscellaneous bacteria, poor monodispersity of micron magnetic beads, etc., to increase the chance of contact, stabilize the reaction solution, and shorten the separation time Effect
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Embodiment 1
[0029] 1. The multi-arm well star polymer-antibody complex is prepared according to the following steps:
[0030] (1) Weigh 10 mg Dobby Star Polymer Dobby Star polyamide-amine, suspend in 4 mL phosphate buffer (PBS, 0.01mol / L, pH 8.0), stir and add 25% of 545 μL of glutaraldehyde aqueous solution, so that the final concentration of glutaraldehyde is 3%. React at room temperature for 3.5 h at the speed of the shaker at 150 r / min;
[0031] (2) Add 0.75 mg of Candida albicans-specific antibody 1 mL dropwise to the above solution to make the final concentration about 3 mg / mL. React at room temperature for 24 h at the speed of the shaker at 150 r / min;
[0032] (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. .
[0033] 2. The long-chain biotin-multi-armed well star polymer-antibody complex is prepared according to the fol...
Embodiment 2
[0039] Example 2 Enrichment effect experiment
[0040] (1) Take 1 mL of concentration as 10 4 cfu / mL Candida albicans 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.
[0041] (2) Enrichment and capture: set up the technical solution group of the present invention (the multi-arm well star polymer group co-modified with Candida albicans antibody and long-chain biotin), and the nanomagnetic The target bacteria were enriched in the bead group and the micron magnetic bead group modified with Candida albicans specific antibody.
[0042] (3) After magnetic separation, pour the supernatant into a sterile centrifuge tube, and wash the separated immunomagnetic beads with Candida albicans twice with PBST, mix well, and wash with 1 mL sterile PBS solution Resuspend the immunomagnetic bead complex.
[0043] (4) Capture rate calculation: After gradient dilution of the enriched t...
Embodiment 3
[0056] Example 3 Enrichment capture experiment
[0057] Conventional magnetic stand separation time is 30min, and all the other are with embodiment 2.
[0058] The catch rate of each group is as follows:
[0059] Capture efficiency of candida albicans-specific antibody-modified micron magnetic bead set Capture efficiency of nano-magnetic beads group modified with specific antibody against Candida albicans Capture Efficiency of Multi-Armed Well Star Polymer Groups Co-modified with Candida albicans Antibody and Long-chain Biotin 55.4% 44.6% 92.1%
[0060] 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 nano-magnetic bead group modified by the Candida albicans specific antibody is the most Obviously, this shows that the capture efficiency of the nano-magnetic bead set...
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