Chemiluminescent enzyme-linked immunoassay method for detecting diclazuril

A technology of chemiluminescent enzyme and diclazuril, which is applied in the direction of analytical materials, measuring devices, scientific instruments, etc., can solve the problems of poor repeatability of results, complicated processing, long time, etc., and achieve the effect of improved sensitivity and high sensitivity

Inactive Publication Date: 2016-05-11
JIANGSU WISE SCI & TECH DEV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of microbiological methods are: time-consuming and lack of specificity
The disadvantages of thin-layer chromatography are: the operation process is complicated and takes a long time; the operators need to undergo professional training; there are many interference factors affecting the analysis, and the repeatability of the results is poor.
The disadvantages of physical and chemical methods such as radioimmunoassay, high performance liquid chromatography, color / mass analysis, and liquid / mass analysis are expensive equipment, complicated sample pretreatment, time-consuming, laborious, difficult to popularize, and high testing costs. In particular, the radioimmunoassay also needs to be equipped with a radioactive source, which has certain risks.

Method used

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  • Chemiluminescent enzyme-linked immunoassay method for detecting diclazuril

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, preparation of immunogen, coated antigen and antibody

[0039] (1) Synthesis of immunogen

[0040] Diclazuril was coupled with bovine serum albumin (BSA) by the p-aminobenzoic acid method to obtain the immunogen. Specifically include the following steps:

[0041] a. Weigh 14mg (100μmol) p-aminobenzoic acid (ABA) and dissolve it in 1.5mL0.2M hydrochloric acid, then weigh 8.3mg (120μmol) of sodium nitrite (NaNO 2 ) was dissolved in 0.24mL of distilled water, stirred at 0-4°C, sodium nitrite (NaNO 2 ) solution was added dropwise to the p-aminobenzoic acid solution, and reacted in the dark for 1 hour to obtain solution A;

[0042] b. Weigh 34mg (100μmol) of diclazuril and dissolve it in 5mL of ice-cold borax buffer (0.05M) (pH8.5, containing 0.15M NaCl), stir at 0-4°C, and add 2mL of the above A solution dropwise Added to the solution, and reacted in the dark for 2 hours to obtain an orange solution;

[0043] c. Add a small amount of boric acid crystals t...

Embodiment 2

[0052] Embodiment 2, the establishment of CL-ELISA detection method

[0053] (1) Optimization of antibody and coated antigen concentration (square matrix)

[0054] Serially dilute each coated antigen longitudinally at 40.0 μg / mL, 20.0 μg / mL, 10.0 μg / mL, 5.0 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.625 μg / mL, 0.3125 μg / mL Coat the microtiter plate with 100 μL / well, place overnight at 0-4°C, wash the plate three times with washing solution, and pat dry each time; block with 250 μL / well blocking solution, place at room temperature for 2 hours, wash the plate three times, and pat dry each time ; Add 100 μL / well of a series of diluted antibodies (1:100 to 1:1024000), place at room temperature for 2.5 hours, wash the plate three times, and pat dry each time; add 100 μL / well of 1:1000 horseradish peroxidase-sheep Anti-rabbit IgG antibody, place at room temperature for 1 hour, wash the plate three times, and pat dry each time; add 100 μL / well of luminescence solution, and measure the luminesc...

Embodiment 3

[0066] Embodiment 3, the chemiluminescent ELISA kit that detects diclazuril

[0067] (1) The composition of the chemiluminescent ELISA kit for detecting diclazuril

[0068] a. A solid phase carrier (elisa plate) coated with a coated antigen (a conjugate of diclazuril and a carrier protein);

[0069] b. Diclazuril standard solution: 0ng / mL, 0.1ng / mL, 0.3ng / mL, 0.9ng / mL, 2.7ng / mL and 8.1ng / mL;

[0070] c. Enzyme-labeled goat anti-rabbit antibody solution: Enzyme-labeled goat anti-rabbit antibody is horseradish peroxidase-goat anti-rabbit IgG stock solution, loaded, and prepared with washing solution to a working concentration of 1:1000 when used;

[0071] d. Diclazuril antibody solution: use the polyclonal antibody prepared by immunizing animals with artificial immune antigens, and dilute the obtained diclazuril antibody with a washing solution to a working concentration of 1:1000;

[0072] e, luminescent solution: use 0.0001M tris(hydroxymethyl)aminomethane solution of p-cres...

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Abstract

The invention discloses a chemiluminescent enzyme-linked immunoassay detection kit for diclazuril. The kit comprises a kit body, an enzyme labeled plate arranged in the kit body and a reagent arranged in the kit body. The kit is characterized in that each hole in the enzyme labeled plate is coated with coating antigen, i.e., diclazuril mother nucleus, and a conjugate of carrier protein. The reagent comprises a diclazuril monoclonal antibody, a horseradish peroxidase-labeled goat anti-mouse antibody, a diclazuril-series standard solution, a concentrated phosphate buffer solution, a concentrated washing solution and a chemiluminescent solution. The chemiluminescent enzyme-linked immunoassay detection kit has the characteristics of high sensitivity, simplicity, rapidness, high accuracy and capacity of detecting a variety of medicines. Compared with traditional colorimetric ELISA methods, the kit provided by the invention enables operation time to be greatly reduced. The kit can be used for detecting residual diclazuril in aquatic products (e.g., fish flesh and shrimp flesh).

Description

technical field [0001] The invention relates to an enzyme-linked immunoassay kit, in particular to a chemiluminescence enzyme-linked immunoassay kit of diclazuril. Background technique [0002] Diclazuril is a triazine benzyl nitrile compound, which is a new type, high efficiency and low toxicity anticoccidiostat, widely used in chicken coccidiosis. The peak period of the main action on coccidia varies with different species of coccidia, such as the main action point on Eimeria tenella is in the sexual cycle of the second generation schizont coccidia. But the giant, Eimeria brucei schizont invalid. The point of action on Eimeria maxima is at the zygote stage of Eimeria brucei; it is highly effective on the microgametophyte stage of Eimeria brucei. Diclozolid also inhibited the formation of sporulated oocysts. Breeders in some countries and regions add diclazuril to food animals, resulting in diclazuril residues in related animal foods. Therefore, regular detection of dicl...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/535
Inventor 洪霞邢海龙江振飞
Owner JIANGSU WISE SCI & TECH DEV
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