Kit for detection of Vibrio parahaemolyticus based on immunomagnetic beads and mno2 nanoparticles
A hemolytic vibrio and nanoparticle technology, applied in the field of microbial detection, can solve the problems of prone to false positives, time-consuming and high requirements
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Embodiment 1
[0023] Example 1 Preparation of Vibrio parahaemolyticus rabbit clone antibody IgG
[0024] Take the concentration as 1×10 9 CFU mL -1 The inactivated bacteria solution was emulsified completely with the same amount of Freund's complete adjuvant / Freund's incomplete adjuvant to prepare the inactivated vaccine. The concentration for the first immunization was 1×10 9 CFU mL -1 Healthy New Zealand white rabbits (provided by the Experimental Animal Center, School of Basic Medicine, Jilin University) were immunized with complete Freund's adjuvant vaccine, and each animal was immunized with 2 mL subcutaneously at multiple points on the back, and the same dose was used in the second and third weeks Rabbits were immunized with Freund's incomplete adjuvant vaccine for the second time and the third time. After 10 days, the immunization was boosted twice with inactivated bacterial solution. Before each immunization, blood was collected from the vein of the rabbit's ear, and the seru...
Embodiment 2
[0027] Example 2 Preparation of Vibrio parahaemolyticus chicken yolk antibody IgY
[0028] Take the concentration as 1×10 9 CFU mL -1 The inactivated bacterial solution was emulsified completely with the same amount of Freund's complete adjuvant / Freund's incomplete adjuvant to prepare the inactivated vaccine, which was used to immunize laying hens. The vaccine was inoculated into chicken breasts by intramuscular multi-point injection. Freund's complete adjuvant vaccine was used for the first immunization, 1 mL per chicken, and the Freund's incomplete adjuvant vaccine was used for the second immunization two weeks later, followed by a booster immunization every two weeks. Eggs before and after immunization were collected and stored in a 4°C refrigerator for later use. The yolk antibody in eggs was extracted by PEG 6000 salting-out method. The titer and specificity of yolk antibody before and after purification were measured by indirect ELISA method. Table 3 is the chicken ...
Embodiment 3
[0031] Example 3 Preparation of Immunomagnetic Beads
[0032]Add 1.08g FeCl 3 .6H 2 O into 22mL ethylene glycol, ultrasonically dissolved for 10min to form a reddish-brown solution; then add 1.2g NaAc and 0.2g sodium citrate to the solution, ultrasonically for 10min to form a dark red solution; finally, add 0.2gPEG6000 to the above solution , ultrasonically mixed, the resulting homogeneous solution was transferred to a reaction kettle, and reacted in an oven at 199°C for 18h. Use a permanent magnet to separate the medium-black solid matter from the reaction solution, wash it alternately with ultrapure water and ethanol for 3-5 times, and obtain Fe 3 o 4 Nanoparticles; take 0.5 mg carboxylated Fe 3 o 4 Nanoparticles, washed twice with PBS, resuspended in 1mL PBS, added 10mg EDC and 10mg NHS to activate the carboxyl group, after 30min, magnetically adsorbed, removed the supernatant, washed 3 times with PBS, resuspended in 1mL PBS, added 50µg anti-parahemolytic Vibrio rabbi...
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