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One-step chemiluminescence/enzyme immunoassay method and kit for detecting residue of malachite green

A chemiluminescent enzyme and malachite green technology, applied in chemiluminescence/bioluminescence, analyzing materials through chemical reactions, and analyzing materials, can solve problems such as long analysis time, narrow detection range, and many operation steps

Active Publication Date: 2013-12-25
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Chinese patent application "a malachite green chemiluminescence enzyme-linked immunoassay method and kit" (application number 201210106196.3 publication date September 12, 2013) adopts a two-step chemiluminescence enzyme-linked immunoassay system and detection method to be tested Detection of malachite green residues in the sample requires many steps, long analysis time and narrow detection range

Method used

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  • One-step chemiluminescence/enzyme immunoassay method and kit for detecting residue of malachite green
  • One-step chemiluminescence/enzyme immunoassay method and kit for detecting residue of malachite green
  • One-step chemiluminescence/enzyme immunoassay method and kit for detecting residue of malachite green

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The preparation of embodiment 1 colorless malachite green monoclonal antibody

[0033] 1.1 Animal immunity

[0034] Using the conjugate of hapten leucomalachite green and carrier protein bovine serum albumin (LMG-BSA) as the immunogen, healthy 6-week-old female BALB / c mice were given basic immunization and 5 booster immunizations, Determination of serum titer and IC 50 , select the mouse with the highest titer and the strongest competition for impact immunization;

[0035] 1.2 Cell fusion and clone screening

[0036] After 3 days of shock immunization, the mice were extirpated after extirpation of eyeballs and bloodletting. The blood was collected, and the spleen was taken aseptically. The immune spleen cells of the mice were fused with Sp2 / 0 cells using cell fusion technology, and the fused cell suspension was added to the previously plated In a 96-well plate with feeder cells, use HAT selection medium to screen for fusion cells;

[0037] When the cells grow to 1 / 1...

Embodiment 2

[0048] Example 2 Establishment of Malachite Green Residue One-Step Chemiluminescent Enzyme Immunoassay Detection Method

[0049] 2.1 Preparation of related reagents

[0050] Carbonate buffer solution (pH9.6, coating solution): Accurately weigh Na 2 CO 3 1.59g, NaHCO 3 2.93g, after dissolving in ultrapure water, adjust the pH to 9.6, and dilute to 1000mL.

[0051] Washing solution (pH7.4): Accurately weigh NaCl8.00g, KH 2 PO 4 0.20g, Na 2 HPO 4 12H 2 O2.90g, KCl0.20g, dissolved in ultrapure water, adjust the pH to 7.4, add Tween-200.50mL, and set the volume to 1000mL.

[0052] Phosphate buffer saline (PBS) (pH7.4): Accurately weigh NaCl8.00g, KH 2 PO 4 0.20g, Na 2 HPO 4 12H 2 O2.90g, KCl0.20g, dissolved in ultrapure water, adjust the pH to 7.4, and set the volume to 1000mL.

[0053] Blocking solution: Accurately weigh 1.0 g of skimmed milk powder, add 20 mL of phosphate buffer, stir evenly until completely dissolved.

[0054] BeyoECL Plus (ultra-sensitive ECL che...

Embodiment 3

[0086] Example 3 Development of Malachite Green Residual One-Step Chemiluminescent Enzyme Immunoassay Detection Kit

[0087] According to the research results of Examples 1 and 2, a malachite green residual one-step chemiluminescent enzyme immunoassay detection kit was assembled.

[0088] 3.1 Kit composition

[0089] A chemiluminescence plate coated with coated antigen (LMG-OVA);

[0090] B colorless malachite green series standard sample solution: 0μg / L, 0.001μg / L, 0.01μg / L, 0.1μg / L, 1μg / L, 10μg / L, 100μg / L;

[0091] C colorless malachite green monoclonal antibody 1H10 working solution;

[0092] D HRP-goat anti-mouse IgG working solution;

[0093] E concentrated washing liquid: NaCl8.00g, KH 2 PO 4 0.20g, Na 2 HPO 4 12H 2 O2.90g, KCl0.20g, Tween-200.50mL, add water to 100mL;

[0094] F Chemiluminescence solution A and B (ultrasensitive ECL chemiluminescence kit P0018--BeyoECL Plus);

[0095] 3.2 Coating of chemiluminescence plate

[0096] Dilute the antigen to 5 μg / ...

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Abstract

The invention discloses a one-step chemiluminescence / enzyme immunoassay method and kit for detecting residue of malachite green. The method combines indirect enzyme immunoreaction and chemiluminescence technologies, simplifies the traditional two-step chemiluminescence / enzyme immunoassay method into a one-step method without additional preparation of an enzyme-labeled monoclonal antibody, and uses 15-35% acetonitrile as a sample dissolving solution based on the characteristic that the 15-35% acetonitrile has limited dissolving power of high-concentration colorless malachite green. The method is used for detecting residue of malachite green in animal-derived foods, has the characteristics of quickness, convenience, specificity, sensitivity, accuracy, wide detection range and the like, better meets the requirements of quick detection, and has favorable application prospects. The IC50 of the kit is 0.45 mu g / L, the recovery rate is 86.37-116.84%, and the accordance rate with a standard detection method is 100%, thus ensuring that the kit is suitable for trace analysis and batch detection of malachite green.

Description

technical field [0001] The invention relates to the field of malachite green residue detection and analysis, in particular to a malachite green residue one-step chemiluminescent enzyme immunoassay detection method and a kit. Background technique [0002] The molecular formula of malachite green (Malachite Green, MG) is C 23 h 25 N 2 Cl, which belongs to triphenylmethane dyes, has been widely used in fish farming because of its remarkable efficacy in the prevention and treatment of saprolegniasis, gill mold, melon worm disease, and fish egg mold. Studies in recent years have found that malachite green, especially its metabolites, has obvious accumulation residues in aquatic animals, and because MG has the hazards of vacuolating animal liver cells, affecting animal growth and reproductive ability, and carcinogenicity, It has seriously threatened aquatic animals and human health, so the United States, Canada, the European Union and many other countries have banned the use of ...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/531G01N21/76
Inventor 吴健敏马玲张启模陈凤莲覃绍敏白安斌韦建兴林俊刘金凤黄红梅关忠谊
Owner GUANGXI VETERINARY RES INST
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