Enzyme linked immunosorbent assay kit of sparfloxacin, establishing method and detecting method of enzyme linked immunosorbent assay kit
An enzyme-linked immunosorbent reagent, sparfloxacin technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of complicated operation process, limited application range, difficult to popularize and apply, etc., and achieve simple processing process, convenient use, Inexpensive effect
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[0044] Example 1 Synthesis of immunogen and preparation of monoclonal antibody
[0045] 1.1 Reagents and instruments
[0046] Sparfloxacin was purchased from Sigma, N,N-dicyclohexylcarbodiimide and N-hydroxysuccinimide were purchased from Shanghai Chemical Reagent Company, bovine serum albumin, chicken ovalbumin, Freund’s complete adjuvant And Freund's incomplete adjuvant comes from Pierce products.
[0047] Bio-Rad imark 680 microplate reader, AE260 electronic balance, purchased from METTLER, Germany; HI9321 acidity meter, HANNA, USA; handheld homogenizer, purchased from IKA, Germany; 93-3 timing constant temperature bidirectional magnetic stirrer, Purchased from Shanghai Yarong Biochemical Instrument Factory.
[0048] 1.2 Synthesis of Sparfloxacin Artificial Antigen
[0049] (1) Weigh 3 mg of sparfloxacin, 3 mg of N-hydroxysuccinimide and 3 mg of N,N-dicyclohexylcarbodiimide and dissolve them in 1 mL of DMF solvent, and react at room temperature for 3 hours to obtain A liquid;
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[0060] Example 2 Establishment of immunoassay method
[0061] 2.1 ELISA method to determine the best coating concentration
[0062] 10μg / mL, 5μg / mL, 2μg / mL, 1μg / mL, 0.5μg / mL, 0.25μg / mL ovalbumin-spafloxacin conjugates were coated with 50μL per well The target plate was coated at 4°C for 2h, washed 4 times, patted dry, added blocking solution, sealed at 4°C for 24h, washed 4 times, patted dry. Join 1:8×10 4 50μL / well of diluted antiserum, incubate at room temperature for 30min, wash 4 times, and immediately add 50μL / well of enzyme-labeled goat anti-mouse antibody. Incubate at room temperature for 30 minutes, wash 4 times, add color developing solution, 50μL / well, and react at room temperature for 15 minutes, add 50μL / well stop solution to stop the reaction, and detect the A value with a microplate reader (450nm). Simultaneously set a blank control well (no antibody, only its dilution) and parallel repeat wells, and the coating concentration with an OD value of about 1.0 is the b...
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[0072] Example 3 Establishment of an enzyme-linked immunoassay kit for detecting sparfloxacin
[0073] An enzyme-linked immunoassay kit for sparfloxacin was constructed to include the following components:
[0074] (1) An ELISA plate coated with sparfloxacin artificially synthesized antigen;
[0075] (2) 2mL, 8×10 4 Times the monoclonal antibody against sparfloxacin;
[0076] (3) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;
[0077] (4) 6 bottles of sparfloxacin standard solution with concentrations of 0μg / L, 10μg / L, 20μg / L, 30μg / L, 50μg / L, 100μg / L;
[0078] (5) Substrate color developer A is carbamide peroxide, and substrate color developer B is tetramethylbenzidine; color developer A and color developer B should be mixed in equal proportions.
[0079] (6) The washing solution is phosphate buffer containing 0.05% Tween-20;
[0080] (7) The concentrated sample diluent is 0.1% Tween-20 phosphate buffer;
[0081] (8) The stop solution is 2mol / L hydrochloric acid soluti...
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