Magnetic particle chemiluminescence kit for detecting sulfanilamide drugs, and applications thereof
A technology for chemiluminescence reagents and sulfonamides, which is applied to a magnetic particle chemiluminescence kit for detecting sulfonamides and its application field, can solve the problems of poor reagent stability, influence of reaction time and temperature, and low false positive rate, etc. Achieve the effect of low detection time, fast detection and high sensitivity
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Embodiment 1
[0037] The preparation of the concrete component of embodiment one kit
[0038] 1) Synthesis of sulfonamide drug hapten
[0039] Preparation of sulfonamide drug haptens by reacting acetaminobenzenesulfonyl chloride with p-aminobenzoic acid.
[0040] 2) Preparation of artificial antigen of sulfa drugs
[0041] The sulfonamide drug hapten and bovine serum albumin are coupled to the immunogen by using the water-soluble carbodiimide method.
[0042] 3) Preparation of monoclonal antibodies
[0043] Animal immunization: Immunize Balb / c mice with the immunogen at a dose of 100 μg / mouse to produce antiserum.
[0044] Cell fusion and cloning: Splenocytes from immunized Balb / c mice were fused with SP2 / 0 myeloma cells at a ratio of 9:1 to obtain hybridoma cell lines of monoclonal antibodies.
[0045]Cell cryopreservation and recovery: the hybridoma cells were made into 1×10 cryopreservation medium 9 cells / ml for long-term storage in liquid nitrogen. When recovering, take out the cr...
Embodiment 2
[0056] The formation of embodiment two kits
[0057] The magnetic particle chemiluminescent detection kit for detecting sulfonamides was assembled so that it contained the following components:
[0058] Fluorescent labeling of FITC-labeled monoclonal antibodies to sulfa drugs
[0059] Luminescent markers of ABEI-labeled sulfonamide drug haptens
[0060] Separation reagent of paramagnetic nanobeads coated with goat anti-FITC monoclonal antibody
[0061] Sulfonamide drug standard solution (0ng / ml, 0.01ng / ml, 0.05ng / ml, 0.25ng / ml, 1.0ng / ml, 5.0ng / ml), the standard diluent is pH7.2, 0.3% azide NaCl, 0.05mol / L Tris buffer. The percentage content is a mass percentage content.
[0062] The concentrations of the sulfonamide quality control solution were 0.02ng / ml and 4.0ng / ml respectively, and the dilution solution of the quality control was pH 7.2, 0.3% sodium azide, and 0.05mol / L Tris buffer. The percentage content is a mass percentage content.
[0063] The concentrated washin...
Embodiment 3
[0064] The detection of sulfa drugs in the actual sample of embodiment three
[0065] 1. Sample pretreatment
[0066] (1) Animal tissue
[0067] High detection limit method (chicken, pork, fish, shrimp)
[0068] Weigh 2.0±0.05g of the homogeneous substance into a 50ml polystyrene centrifuge tube, add 8ml of ethyl acetate, shake immediately with a shaker for 5min, and centrifuge for 5min above 3000g; take 4ml of the supernatant into a 10ml clean glass test tube, and place Blow dry under nitrogen flow in a water bath at ~60°C; add 1ml of n-hexane, vortex for 30s with a vortexer to dissolve the dry residue, add 1ml of complex solution working solution, vortex for 30s with a vortexer, then transfer to a 2ml centrifuge tube, over 3000g , centrifuge at room temperature (20-25° C.) for 5 min; remove the upper n-hexane phase, and take 50 μl of the lower aqueous phase for analysis, sample dilution factor: 1.
[0069] (2) Egg pretreatment method
[0070] Homogenize the egg sample wi...
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