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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, etc.

Active Publication Date: 2014-08-20
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 samples of chemiluminescence enzyme-linked immunoassay kit

[0080] (1) The preparation of lotion: KH 2 PO 4 0.4g, Na 2 HPO 4 ·12H 2 O 5.8g, NaCl 16g, KCl0.4g, Tween-20 0.05% by volume 1mL, add distilled water to 2000mL to prepare a PBST phosphate buffer with a pH of 7.4.

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

[0082] (3) Preparation of luminescent substrate solution: 2.0 mg luminol and 0.8 mg 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 the luminescent plate m...

Embodiment 2

[0089] Example 2 Detection method of chemiluminescence enzyme-linked immunoassay kit

[0090] (1) Take the kit out of the refrigerated environment, put it at room temperature (20~24℃) to equilibrate for more than 30 minutes, fix enough strips for the standard and sample to the bracket, do two parallel experiments for the standard and sample, in order Numbering.

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

[0092] (3) Pour out the liquid in the hole, put the microwell holder upside down on absorbent paper and tap (3 times per round of plate washing) to ensure that the liquid in the hole is completely removed. Fill the well with 250 μL of washing solution, pour out the liquid in the well again, and repeat the operation 5 times.

[0093] (4) Add 100μL of enzyme-labeled antibody ...

Embodiment 3

[0099] Example 3 Detection of fish meat samples

[0100] Chop the negative and positive fish samples (concentration confirmed by HPLC), take 5.0 g of the homogenized fish, add 5 mL of the extract in the kit, homogenize at high speed for 10 seconds, add 5.0 mL of acetonitrile, shake and extract for 10 minutes, then centrifuge at 4000r Centrifuge for 5 min at / min, transfer the supernatant to a new centrifuge tube; add 5.0 mL of acetonitrile to the residue, shake the sample for 5 min, then centrifuge at 4000r / min for 5 min at room temperature, take the supernatant and combine it for the first time Supernatant in the tube. After mixing all the supernatants, add 10 mL of dichloromethane and shake vigorously for 5 seconds, then remove the lower layer solution and transfer to a rotary evaporator, repeat the above operation with 10 mL of dichloromethane; combine the lower layer solutions at 40°C and rotate to dryness , Use 0.5mL acetonitrile for reconstitution, add 4.5mL PBS buffer sol...

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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 present invention relates to the technical field of chemiluminescence enzyme-linked immunoassay, in particular to a chemiluminescence enzyme-linked immunoassay method for malachite green and a kit for detecting malachite green using the method. Background technique [0002] Malachite green (malachite, MG), also known as basic green and base block green, is a triphenylmethane compound. It has high toxin, high residue, high carcinogenic, high teratogenic, mutagenic and other side effects, and is harmful to human health. The environment causes serious harm. The FDA has long banned the use of malachite green in fisheries. The United States, Canada, and the European Union have stipulated that malachite green should not be detected in aquatic products such as edible fish. my country listed it in the "Prohibited Veterinary Drugs and Their Products for Food Animals" in May 2002. Compound List" (Announcement No. 193 of the Ministry of Agriculture). However, due...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/76
Inventor 曾道平路俊山
Owner 广州万联生物科技有限公司
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