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Malachite chemiluminescence ELISA detection method and kit

An immunodetection method, chemiluminescent enzyme technology, used in chemiluminescence/bioluminescence, analysis by chemically reacting materials, measurement devices, etc.

Active Publication Date: 2012-09-12
广州万联生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to aim at the deficiencies of the existing kits used for malachite green detection, to provide a method for the detection of malachite green chemiluminescent ELISA with high specificity and high sensitivity.

Method used

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  • Malachite chemiluminescence ELISA detection method and kit
  • Malachite chemiluminescence ELISA detection method and kit
  • Malachite chemiluminescence ELISA detection method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1 Preparation of Chemiluminescent ELISA Kit Samples

[0080] (1) Preparation of lotion: mix KH 2 PO 4 0.4g, Na 2 HPO 4 12H 2 O 5.8g, NaCl 16g, KCl 0.4g, Tween-20 0.05% by volume 1mL, add distilled water to 2000mL to prepare PBST phosphate buffer saline with a pH value of 7.4.

[0081](2) Preparation of blocking solution: 1.0-5.0 g of skimmed milk powder was dissolved in 100 mL of distilled water.

[0082] (3) Preparation of luminescent substrate solution: 2.0 mg of luminol and 0.8 mg of p-iodophenol were dissolved in 10 mL of Tris-HCl buffer (pH 9.0), and stored at 4°C.

[0083] (4) Preparation of substrate buffer: 20 μL of hydrogen peroxide (30%) was dissolved in 10 mL of Tris-HCl buffer (pH 7.0), and stored at 4°C.

[0084] (5) Preparation of extract: 10mL 0.36mol / L hydroxylamine hydrochloride solution, 15mL 1.0mol / L p-toluenesulfonic acid solution and 25mL 0.05mol / L ammonium acetate buffer, mix well, and store at 4°C.

[0085] (6) Coating of luminesce...

Embodiment 2

[0089] Example 2 Detection method of chemiluminescent ELISA kit

[0090] (1) Take the kit out of the refrigerated environment, place it at room temperature (20-24°C) to equilibrate for more than 30 minutes, fix enough strips for standards and samples on the bracket, and do two parallel experiments for standards and samples, in order serial number.

[0091] (2) Add 50 μL of standard substance to the standard well, and add 50 μL of the sample to be tested into the sample well. Then add 50 μL of anti-malachite green antibody solution to each well, and pat to mix. Incubate at room temperature for 30 min with shaking.

[0092] (3) Pour out the liquid in the well, turn the microwell frame upside down on the absorbent paper and pat (3 times for each round of plate washing) to ensure that the liquid in the well is completely removed. Fill the wells with 250 μL of washing solution, pour off the liquid in the microwells again, and repeat the operation 5 times.

[0093] (4) Add 100 μ...

Embodiment 3

[0099] Example 3 Detection of fish samples

[0100] Mince the negative and positive fish samples (concentration confirmed by HPLC), take 5.0g of homogenized fish, add 5mL of the extraction solution in the kit, add 5.0mL of acetonitrile after high-speed homogenization for 10s, shake and extract for 10min, and then centrifuge for 4000r / min centrifuge for 5min, transfer the supernatant to a new centrifuge tube; add 5.0mL acetonitrile to the residue, shake the sample for 5min, then centrifuge at 4000r / min at room temperature for 5min, take the supernatant and combine it into the first in the supernatant tube. After mixing all the supernatants, add 10 mL of dichloromethane and shake vigorously for 5 seconds, then transfer the solution from the lower layer to a rotary evaporating flask, and repeat the above operation once with 10 mL of dichloromethane; combine the solutions of the lower layers and evaporate to dryness at 40°C , redissolve with 0.5mL acetonitrile, add 4.5mL PBS buf...

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Abstract

The invention discloses a malachite chemiluminescence ELISA detection method and a kit. According to the invention, it is the first time to discuss chemiluminescence ELISA mechanism of malachite, a malachitechemiluminescence ELISA detection system and a detection method are successively established, and perfect combination of high sensitivity and high specificity is realized. The kit provided by the invention has high sensitivity, accuracy, precision and stability, is used to simplify operation steps and reaction time and reduce errors caused by complex operation, is very suitable for trace analysis and batch detection of malachite residues, and is of great practical application significance.

Description

technical field [0001] The invention relates to the technical field of chemiluminescence ELISA detection, in particular to a chemiluminescence ELISA detection method for malachite green and a kit for detecting malachite green by using the method. Background technique [0002] Malachite green (malachite, MG), also known as basic green, base block green, belongs to triphenylmethane compounds, has high toxicity, high residue, high carcinogenicity, high teratogenicity, mutagenicity and other side effects, which are harmful to human health and serious harm to the environment. The FDA has already prohibited the use of malachite green in fisheries. The United States, Canada, and the European Union all stipulate that malachite green must not be detected in edible fish and other aquatic products. Compound List" (Announcement No. 193 of the Ministry of Agriculture). However, due to the low price of malachite green and the lack of good substitutes for saprolegniasis prevention, some ...

Claims

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Application Information

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IPC IPC(8): G01N21/76G01N33/543G01N33/577
Inventor 曾道平路俊山
Owner 广州万联生物科技有限公司
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