Method for magnetically separating yersinia enterocolitica (YE)
A technology of enterocolitis and Yersinia, which is applied in the field of isolation of food-borne pathogenic bacteria, can solve the problems of separation failure, antibody spatial conformation change, poor monodispersity of micron magnetic beads, etc., to increase the chance of contact, Effects of improving separation efficiency and shortening separation time
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Embodiment 1
[0032] 1. The dendrimer-antibody complex is prepared according to the following steps:
[0033] (1) Weigh 1.1 mg of aminated dendrimers, suspend in 4 mL of phosphate buffered saline (PBS, 0.01mol / L, pH 8.0), stir and add 545 μL of 25% glutaraldehyde aqueous solution dropwise to make The final concentration of dialdehyde was 3%. React at room temperature for 3.5 h at a rotating speed of 150 r / min on a shaker;
[0034] (2) Add Yersinia enterocolitica dropwise to the above solution YE 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;
[0035] (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.
[0036] 2. The long-chain biotin-dendrimer-antibody complex is prepared according to the following steps:
[0037] (1) Su...
Embodiment 2
[0042] Example 2 Enrichment effect experiment
[0043] (1) Take 1 mL of concentration as 10 4 cfu / mL YE Centrifuge at 12,000 rpm for 5 min in a 1.5 mL sterile centrifuge tube, discard the supernatant, and resuspend with an equal volume of sterile PBS solution.
[0044] (2) Enrichment and capture: respectively set the technical solution group of the present invention ( YE dendrimers co-modified with antibodies and long-chain biotin), YE Specific antibody-modified nano-magnetic bead set, YE Specific antibody-modified micron magnetic bead group enriches target bacteria.
[0045] (3) After magnetic separation, pour the supernatant into a sterile centrifuge tube, and capture the YE The immunomagnetic beads were washed twice with PBST, mixed well, and the immunomagnetic bead complex was resuspended with 1 mL sterile PBS solution.
[0046] (4) Capture rate calculation: After gradient dilution of the enriched target bacteria resuspension in each group, count each gradient ...
Embodiment 3
[0058] Example 3 Enrichment capture experiment
[0059] Conventional magnetic stand separation time is 30 min, all the other are with embodiment 2.
[0060] YE Specific antibody-modified micron magnetic beads Obtaining rate YE Capture efficiency of specific antibody-modified nanomagnetic bead sets YE Capture efficiency of dendrimers co-modified with antibodies and long-chain biotin 63.7% 38.6% 93.8%
[0061] Experimental result shows, separates 3 min among the comparative example 2, and when separation time reaches 30 min, the capture efficiency of three groups has all been improved, especially YE The capture efficiency of the specific antibody-modified nano-magnetic bead group is the most obvious, which shows that the capture efficiency of the nano-magnetic bead group can be greatly improved by extending the time, but it is still lower than the short-time separation (3 min) YE Capture efficiency of dendrimers co-modified with antibodies and...
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