Direct competition method ELISA reagent box for detecting melamine
An enzyme-linked immunosorbent reagent and melamine technology, which is applied in the direction of measuring devices, instruments, and material analysis through optical means, to achieve the effects of low equipment requirements, long storage time of reagents, and high accuracy
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Embodiment 1 3
[0023] Synthesis of embodiment 1 melamine immunogen and preparation of immune serum
[0024] 1.1 Reagents and instruments
[0025] Melamine (Beijing Bailingwei Reagent Co., Ltd.), succinic anhydride (purchased from Beijing Yaobei Biotechnology Co., Ltd.), pyridine (Pyridine, AR.WM=79.10, content > 99.5%, Tianjin Kemiou Chemical Reagent Development Center), N , N-dimethylformamide (Dimethylformamide, DMF, produced by ACROSORGANICS, New Jersey, U.S., purchased from Bailingwei Chemical Reagent Company), tributylamine (Tributylamine, produced by ACROSORGANICS, New Jersey, U.S., purchased from Bailingwei Chemical Reagent Company), chlorine Isobutyl formate was purchased (from Bailingwei Chemical Reagent Company), bovine serum albumin (BSA), horseradish peroxidase (HRP), etc. (purchased from Beijing Yaobei Biotechnology Co., Ltd.), and other reagents were of analytical grade.
[0026] Double-beam ultraviolet-visible spectrophotometer (TU-1909, Beijing Puxi General Instrument Co., L...
Embodiment 2
[0034] Embodiment 2 The establishment of immunoassay method
[0035] 2.1 ELISA matrix method to determine the optimal reaction concentration
[0036] Coat the microtiter plate with 100 μl per well of melamine antibody at serial concentrations of 1000 μg / ml, 100 μg / ml, 10 μg / ml, 5 μg / ml, 1 μg / ml, and 0.25 μg / ml, coat overnight at 4°C, and wash 3 times. Pat dry, block overnight at 4°C with 200 μl blocking solution per well, wash 3 times, and pat dry. Add enzyme-labeled melamine diluted from 1:200, react at room temperature for 30 minutes, wash three times, add 50 μl of substrate A solution and 50 μl of substrate B solution, protect from light at room temperature for 15 minutes, stop the reaction with 50 μl of stop solution, and use a microplate reader A value (450nm) was detected. Set two parallels at the same time, and take the coating concentration when the OD value is about 1.0 as the optimal concentration. The test data are listed in Table 1.
[0037] Table 1 OD value of...
Embodiment 3
[0045] Embodiment 3 detects the formation of the ELISA kit of melamine
[0046] Set up the enzyme-linked immunosorbent assay kit that detects melamine, make it comprise the following components:
[0047] (1) ELISA plate coated with melamine antibody;
[0048] (2) Enzyme-labeled melamine whose concentration is 1 mg / L;
[0049] (3) 6 bottles of melamine standard solution, the concentrations are 0μg / L, 1μg / L, 5μg / L, 10μg / L, 50μg / L, 100μg / L;
[0050] (4) Substrate chromogenic solution A liquid is carbamide peroxide, and substrate chromogenic solution B liquid is tetramethylbenzidine;
[0051] (6) The washing liquid is a phosphate buffer containing 0.05% Tween 20;
[0052] (7) The concentrated sample diluent is a phosphate buffered saline solution of 0.1% Tween-20;
[0053] (8) The enzyme-labeled melamine dilution is a phosphate buffer containing 0.01% Tween-20;
[0054] (9) The stop solution is 2mol / L hydrochloric acid solution.
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