Chemiluminesent immunoassay kit for detecting fipronil in tea
A chemiluminescence immunoassay and detection kit technology, applied in the field of immunological detection, can solve the problems of strong carcinogenicity and high toxicity of fipronil, and achieve the effects of simple instrument, reduction of use links, and guarantee of long-term effectiveness
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Embodiment 1
[0040] 1. Preparation of each component of the kit
[0041] (1) Preparation of fipronil hapten: acidify fipronil, react with sodium nitrite in a low-temperature environment without light at 4°C, and generate intermediates containing diazonium cations. The diazotized fipronil was used as a hapten for subsequent synthesis of immune antigens and coating antigens.
[0042] (2) Preparation of fipronil-bovine serum albumin (BSA) immunogen: Fipronil and bovine serum albumin (BSA) were coupled by diazotization method to obtain immune antigen.
[0043] Preparation of fipronil-oval serum albumin (OVA) coated antigen: coupling fipronil and egg serum albumin (OVA) by diazotization method to obtain coated antigen.
[0044]Preparation of fipronil-peroxidase-labeled antibody: For 6-8 week-old female BALB / c mice (body weight 18-20g), the high-dose immunization scheme is as follows: 160 µg fipronil-BSA and Mix an equal amount of complete Freund's adjuvant and inject subcutaneously. After 3 ...
Embodiment 2
[0073] The chemiluminescent immunoassay kit for detecting fipronil includes the following components:
[0074] (1) A 96-well opaque white microplate (8 wells x 12 strips) is coated with fipronil-mouse serum albumin conjugate, and is vacuum-sealed in an aluminum foil bag.
[0075] (2) 6 bottles of fipronil standard solution, the concentrations are:
[0076] 0 ng / mL, 0.02 ng / mL, 0.08 ng / mL, 0.32 ng / mL, 1.28 ng / mL, 5.12 ng / mL
[0077] (3) Fipronil-horseradish peroxidase labeled antibody solution.
[0078] (4) Luminescence substrate A solution (luminol and enhancer), luminescence substrate B solution (urea hydrogen peroxide).
[0079] (5) 2 times concentrated sample diluent. Before use, please dilute 1:1 (1 part concentrated diluent + 1 part deionized water) to become the working sample diluent, and the diluted working sample diluent is 0.05 mol / L, pH 7.4 Tris-HCl buffer.
[0080] (6) 20 times concentrated washing solution. Before use, please dilute at 1:19 (1 part of concent...
Embodiment 3
[0082] The chemiluminescent immunoassay kit for detecting fipronil includes the following components:
[0083] (1) A 96-well opaque white microplate (8 wells x 12 strips) is coated with fipronil-egg albumin conjugate, and is vacuum-sealed in an aluminum foil bag.
[0084] (2) 6 bottles of fipronil standard solution, the concentrations are:
[0085] 0 ng / mL, 0.02 ng / mL, 0.08 ng / mL, 0.32 ng / mL, 1.28 ng / mL, 5.12 ng / mL
[0086] (3) Fipronil-horseradish peroxidase labeled antibody solution.
[0087] (4) Luminescence substrate A solution (luminol and enhancer), luminescence substrate B solution (urea hydrogen peroxide).
[0088] (5) 2 times concentrated sample diluent. Before use, please dilute 1:1 (1 part of concentrated diluent + 1 part of deionized water) to become the working sample diluent, and the diluted working sample diluent is 0.05 mol / L, pH 7.4 glycine-HCl buffer.
[0089] (6) 20 times concentrated washing solution. Before use, please dilute at 1:19 (1 part of concen...
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