One-step homogeneous chemiluminescent detection method for micromolecule and particle used therein

A homogeneous chemiluminescence, small molecule technology, applied in the direction of chemical reaction of materials for analysis, chemiluminescence/bioluminescence, measurement devices, etc. expensive, etc.

Inactive Publication Date: 2015-09-09
HANGZHOU JINXI BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method also has its disadvantages: ① the sensitivity is often low when detecting biological samples (the sensitivity is generally only 1ng/ml)
When detecting blood and urine, due to the matrix effect of the sample and the substances in the sample that absorb the light emitted by the luminescent particles, the detection sensitivity is not as high as in reaction solutions such as PBS; ②The substances in the acceptor particles ar

Method used

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  • One-step homogeneous chemiluminescent detection method for micromolecule and particle used therein
  • One-step homogeneous chemiluminescent detection method for micromolecule and particle used therein
  • One-step homogeneous chemiluminescent detection method for micromolecule and particle used therein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1-1

[0090] Example 1-1. Preparation of clenbuterol-BSA-labeled homogeneous chemiluminescence acceptor particles and mouse-resistant clenbuterol homogeneous chemiluminescence donor particles for one-step homogeneous chemiluminescence detection; Do the following steps:

[0091] 1. Preparation of clenbuterol-BSA-labeled homogeneous chemiluminescent acceptor particles

[0092] ①. Label the BSA carrier protein with clenbuterol small molecule:

[0093] Firstly, the diazonium method is used to couple the veterinary small molecule clenbuterol and the carrier protein-bovine serum albumin (BSA) at a certain molar ratio, and dialyze with PBS to obtain the carrier protein labeled with the small molecule. Specifically:

[0094] Dissolve 5mg (18.04umol) of clenbuterol hydrochloride in 1ml of pre-cooled dilute hydrochloric acid (pH=3.0) at 3-8°C, add 10mg of nitrite---sodium nitrite (that is, nitrite: Clenbuterol The mass ratio of Tero is 2:1), reacted at room temperature for 6 hours, and the...

Embodiment 1-2

[0111] Embodiment 1-2, detection

[0112]Prepare assay buffer: In 25 mM HEPES pH 7.4, add 2 mg / mL Dextran-500, 50 mmol / L NaCl, 0.5% (w / v) BSA, 0.02% (w / v) bovine gama protein, 0.1% ( v / v) Tween 20, 0.01% (v / v) Proclin-300, fully dissolved, filtered and stored at 4°C.

[0113] That is, the preparation method of the above analysis buffer is as follows: in every 100ml of 25mM HEPES with pH7.4, add 200mg of Dextran-500, 5mmol of NaCl, 0.5g of BSA, 0.02g of bovine gama protein, 0.1ml of Warm 20, 0.01ml of Proclin-300, fully dissolved, filter and store at 4°C.

[0114] Animal tissue sample pretreatment:

[0115] Animal tissue processing to obtain clenbuterol: the sample can be processed by liquid-liquid extraction method, first adjust the pH value of the tissue to above pKa 8.0 of clenbuterol, so that it is released as a single molecule, and then extracted with an organic solvent, This method recovered more than 80% of the clenbuterol samples.

[0116] Remarks: The above-mention...

Embodiment 1-3

[0123] Embodiment 1-3, verification test of shelf life:

[0124] Such as Figure 5 , will (ATTA-Eu 3+ ) coated microparticles and donor microparticles are labeled with clenbuterol corresponding antibodies or antigens (that is, clenbuterol-BSA-labeled homogeneous chemiluminescent acceptor microparticles, conjugated anti-gram Homogeneous chemiluminescent donor microparticles of Renbutrol mouse resistance), plus microparticle protectants (specifically 20% (w / v) glucose 20ul, 10% (w / v) mannitol 20ul, 0.5% (w / v) ) BSA 20ul, 0.01% (v / v) Proclin-300 20ul) until the concentration is 5mg / mL, after freeze-drying together, take out a copy of Clent every month for testing the gradient dilution of PBS buffer (PH=7.4) Luo standard product, get 12 months continuously (taking the average value), test result is as follows Figure 5 As shown, the detection sensitivity did not change much.

[0125] The preparation method of the above-mentioned microparticle protectant is as follows: 20ul of ...

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Abstract

The invention discloses a particle used for detection of a veterinary drug micromolecule. The particle comprises a receptor particle and a donor particle and is prepared through the following steps: marking carrier protein with a veterinary drug molecule; coupling the veterinary drug molecule-marked veterinary drug with a homogeneous chemiluminescent receptor ball; and preparing a homogeneous chemiluminescent receptor ball coupled with a veterinary drug molecule antibody. The invention further discloses a one-step homogeneous chemiluminescent method for detection of the micromolecule with the particle. The one-step homogeneous chemiluminescent method is used for homogeneous, rapid and high-sensitivity detection of veterinary drug residue and for testing of the contents of frequently used veterinary drug components in feeds, e.g., clenbuterol, ractopamine, salbutamol and terbutaline.

Description

technical field [0001] The invention relates to a one-step homogeneous chemiluminescence method for small molecule detection and microparticles used (including acceptor microparticles and donor microparticles). Background technique [0002] The large-scale and integrated production of modern animal husbandry is becoming more and more mature. In order to ensure the rapid and healthy growth of livestock and poultry, more and more veterinary drugs are used in actual breeding, but unreasonable use of veterinary drugs often results in serious veterinary drug residues. The quality of life has brought profound social problems. According to the regulations of the WHO organization, veterinary drug residues refer to the original veterinary drug, its metabolites and associated impurities contained in any edible part of animal products. According to reports, there are more than 120 types of veterinary drug residues, and beta-stimulants (beta-adrenergic receptor blockers) are one of the...

Claims

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Application Information

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IPC IPC(8): G01N21/76G01N33/543
Inventor 朱成钢向闯南江学成易文
Owner HANGZHOU JINXI BIOLOGICAL TECH
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