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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

Active Publication Date: 2013-12-11
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the commonly used physical and chemical analysis methods require expensive instruments and equipment, skilled professionals and other conditions, and the operation process is complicated and the detection time is long, so the scope of application is limited, and it is difficult to popularize and apply in actual production

Method used

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  • Enzyme linked immunosorbent assay kit of sparfloxacin, establishing method and detecting method of enzyme linked immunosorbent assay kit
  • Enzyme linked immunosorbent assay kit of sparfloxacin, establishing method and detecting method of enzyme linked immunosorbent assay kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[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 from Pierce products.

[0047] Bio-Rad imark 680 microplate reader, AE260 electronic balance, purchased from Germany METTLER company; HI9321 acidity meter, American HANNA company; hand-held homogenizer, purchased from Germany IKA company; 93-3 time constant temperature two-way 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, dissolve them in 1 mL of DMF solvent, and react at room temperature for 3 hours to ob...

Embodiment 2

[0060] Example 2 Establishment of immunoassay method

[0061] 2.1 ELISA method to determine the optimal coating concentration

[0062] Coat the enzyme with ovalbumin-sparfloxacin conjugate at a concentration of 10 μg / mL, 5 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL in an amount of 50 μL per well The target plate was coated at 4°C for 2 hours, washed 4 times, patted dry, added blocking solution, blocked at 4°C for 24 hours, washed 4 times, and patted dry. Join 1:8×10 4 50 μL / well of diluted antiserum was incubated at room temperature for 30 min, washed 4 times, and 50 μL / well of enzyme-labeled goat anti-mouse antibody was immediately added. React at room temperature for 30 minutes, wash 4 times, add chromogenic solution, 50 μL / well, chromogenic reaction at room temperature for 15 minutes, add 50 μL / well stop solution to terminate the reaction, and detect the A value with a microplate reader (450 nm). At the same time, set blank control wells (no antibody, only add it...

Embodiment 3

[0072] Example 3 Construction of the enzyme-linked immunosorbent assay kit for detecting sparfloxacin

[0073] Construct the ELISA kit of sparfloxacin to include the following components:

[0074] (1) ELISA plate coated with sparfloxacin artificially synthesized antigen;

[0075] (2) 2mL, 8×10 4 Times anti-sparfloxacin monoclonal antibody;

[0076] (3) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0077] (4) 6 bottles of sparfloxacin standard solution, the concentrations are 0μg / L, 10μg / L, 20μg / L, 30μg / L, 50μg / L, 100μg / L;

[0078] (5) Substrate chromogenic solution A is carbamide peroxide, and substrate chromogenic solution B is tetramethylbenzidine; chromogenic agent A and chromogenic agent B should be mixed in equal proportions.

[0079] (6) The washing solution is a phosphate buffer containing 0.05% Tween-20;

[0080] (7) The concentrated sample diluent is a phosphate buffered saline solution of 0.1% Tween-20;

[0081] (8) The stop solution is 2...

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Abstract

The invention discloses an enzyme linked immunosorbent assay kit for detecting sparfloxacin, which comprises a kit body, wherein the kit body is internally provided with a sparfloxacin specific antibody, a conjugate and an enzyme labeled second antibody of the sparfloxacin and carrier protein, a sparfloxacin antigen or specific antibody coated plate, a sparfloxacin standard solution, a substrate development solution, a cleaning solution and a stop solution, and the sparfloxacin specific antibody is a sparfloxacin monoclonal antibody. Main reagents in the enzyme linked immunosorbent assay kit of the sparfloxacin are provided in a working solution form, and the enzyme linked immunosorbent assay kit is convenient to use and low in price, has low requirements on pretreatment of a sample during detection, is simple in treatment process, can be used for simultaneously and quickly screening massive samples, and has the characteristics of quickness, simplicity, convenience, accuracy, high sensitivity and the like in comparison to an instrumental analysis technique.

Description

technical field [0001] The invention relates to the fields of ELISA and food additive residue detection. In particular, it relates to an enzyme-linked immunosorbent assay kit for detection of sparfloxacin residues in food and its composition and detection method. Background technique [0002] Sparfloxacin (SPFX), molecular formula C 19 h 22 f 2 N 4 o 3 ; Relative molecular mass 392.41; Melting point 137-141°C; Appearance is yellow crystal or crystalline powder; Fat-soluble, slightly soluble in glacial acetic acid, chloroform, slightly soluble in methanol, ethanol, almost insoluble in ether and water; Stablize. SPFX is widely used in animal husbandry, aquaculture, veterinary and other industries in my country, but it has certain toxic and side effects, mainly including gastrointestinal reactions, central nervous system toxicity and phototoxicity. In recent years, drug residues and drug resistance in animal food caused by improper use, as well as water and soil pollutio...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/535
Inventor 邓瑞广张改平胡骁飞柴书军杨继飞贾国超
Owner HENAN ACAD OF AGRI SCI
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