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Enzyme linked immunosorbent assay kit for detecting bidens bipinnata cyclic peptide, and preparation and application thereof

An enzyme-linked immunosorbent reagent and a technology for phalloidin, which is applied to a kit for detecting phalloidin and the field of preparation thereof, can solve the problems of false positives, expensive instruments, low sensitivity and the like, and achieve strong specificity and high accuracy. , good stability

Active Publication Date: 2020-06-12
北京维德维康生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing methods have problems such as low detection throughput, false positives, low sensitivity, and severe matrix effects. Methods with strong specificity and high sensitivity are cumbersome to operate and expensive instruments, and are not suitable for screening and detection of large batches of samples, and cannot meet the needs of on-site detection. need

Method used

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  • Enzyme linked immunosorbent assay kit for detecting bidens bipinnata cyclic peptide, and preparation and application thereof
  • Enzyme linked immunosorbent assay kit for detecting bidens bipinnata cyclic peptide, and preparation and application thereof
  • Enzyme linked immunosorbent assay kit for detecting bidens bipinnata cyclic peptide, and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1. Preparation of an enzyme-linked immunosorbent assay kit for detecting phalloidin

[0051] The kit is composed as follows: ELISA plate (coated with a conjugate of phalloidin drug and carrier protein), antibody working solution (containing phalloidin drug monoclonal antibody), enzyme marker working solution (containing horseradish Peroxidase-labeled anti-phalloidin drug monoclonal antibody anti-antibody), washing solution, sample diluent, sample extraction solution, working solution containing different gradient concentrations of phalloidin standard, substrate chromogenic solution, stop solution.

[0052] The preparation method is as follows:

[0053] 1. Preparation of ELISA plate

[0054] 1. Preparation of Phalloidin Coating Source

[0055] (1) Preparation of phalloidin hapten

[0056] Rinse the 50ml round bottom flask, dry it with ethanol, wrap it with tinfoil to prevent it from being exposed to light, fix it on the stirrer, and add a stirring bar. Weigh...

Embodiment 2

[0099] The using method of embodiment 2, embodiment 1 kit

[0100] 1. Sample pretreatment

[0101] 1. Pretreatment of serum and urine samples

[0102] Accurately weigh 1 ± 0.01 g (or mL) of sample and add 5 mL of sample extract, at room temperature (25 ± 2°C), vortex at high speed for 1 min, and centrifuge at 4000 g for 5 min to obtain a sample solution. Take 200 μL sample solution and add 200 μL sample diluent, vortex at high speed for 1 min, and take 50 μL supernatant for analysis.

[0103] Two, use embodiment 1 kit to detect

[0104] 1. Take the microplate and insert it into the microplate rack, and record the position of each standard and sample, make 3 parallels for each sample, seal the unused microplate strip with a ziplock bag, and store it in a 2- 8°C environment;

[0105] 2. Add 50 μL of each standard working solution or sample solution into the corresponding standard or sample well;

[0106] 3. Add 50 μL antibody working solution to each plate well;

[0107] 4...

Embodiment 3

[0128] Specificity, detection limit, accuracy, precision detection of embodiment 3, embodiment 1 kit

[0129] 1. Specificity test of the kit:

[0130] The specificity of the phalloidin ELISA kit is determined by cross-reactivity tests with the corresponding substances.

[0131] Make serial dilutions of phalloidin and its analogs (α-amanitin, β-amanitin), respectively, and perform operations according to Example 2, and use the serial dilutions of phalloidin and its analogs Replace the "Phalloidin Standard Working Solution" in it, make a standard curve, and find out the respective 50% inhibitory concentrations (IC 50 ), the specific method is as follows: get the phalloidin concentration (μg / L) corresponding to the ordinate value equal to 50%, that is, IC 50 value. Use the following formula to calculate the cross-reactivity rate of the kit to phalloidin and various commonly used rodenticides:

[0132] Cross-reactivity rate (%)=(phalloidin concentration causing 50% inhibition / ...

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Abstract

The invention discloses an enzyme linked immunosorbent assay kit for detecting bidens bipinnata cyclic peptide, and preparation and application thereof. The kit provided by the invention is composed of an elisa plate (coated with a conjugate of a bidens bipinnata cyclopeptide drug hapten shown as a formula I and carrier protein), an antibody working solution (containing a sticktight cyclic peptidedrug monoclonal antibody), an enzyme marker working solution (containing an anti-antibody of a horseradish peroxidase marked anti-sticktight cyclic peptide drug monoclonal antibody), a washing solution, a sample diluent, a sample extracting solution, a standard substance solution containing sticktight cyclic peptides with different gradient concentrations, a substrate developing solution and a stop solution. The bidens bipinnata cyclic peptide detection kit provided by the invention can be used for detecting bidens bipinnata cyclic peptide in shellfish food, the detection limit of the bidensbipinnata cyclic peptide in serum and urine can be set to be 1.0 microgram / L, the inter-batch variation coefficient is smaller than 10%, the intra-batch variation coefficient is smaller than 15%, andthe enzyme linked immunosorbent assay kit for detecting bidens bipinnata cyclic peptide, and preparation and application thereof have the advantages of being easy and rapid to operate, high in sensitivity, high in specificity and high in accuracy and has great value in rapid detection of food poisoning.

Description

technical field [0001] The invention belongs to the field of rapid detection of drug residues, and relates to a kit for detecting phalloidin, a preparation method and application thereof. Background technique [0002] Mushroom food poisoning has always been one of the food safety issues that many countries in the world have focused on. For many years, mushroom poisoning has also been the main cause of death caused by food poisoning accidents in my country. Phalloidin belongs to bicyclic polypeptide compounds, and exists in some wild fungi of Amanita, Galerina, and Lepiota. Food poisoning caused by phalloidin is easy Misdiagnosis and lack of targeted treatment, so early diagnosis is particularly important for the treatment of poisoning. [0003] At present, the detection methods of phalloidin toxoids in biological samples such as plasma and urine mainly include capillary electrophoresis, radioimmunoassay, high performance liquid chromatography and mass spectrometry, among wh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/58G01N33/577G01N33/543
CPCG01N33/6893G01N33/581G01N33/577G01N33/54393G01N2800/709G01N2333/375
Inventor 马立才刘河冰温凯杨柳丁亚芳崔乃元聂靖东邢维维刘薇
Owner 北京维德维康生物技术有限公司
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